Patent Application
20210277052
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 1, 2021, is named SequenceListing3102632US02 ST25.txt and is 321,853 bytes in size.
This application is generally directed to the field of molecular biology and more specifically to methodology for using the Grp94 Pre-N Domain to regulate N-linked glycosylation in order to preserve chaperone function.
U.S. Pat. No. 7,589,174 to Argon et al., titled “GRP94-BASED COMPOSITIONS AND METHODS OF USE THEREOF,” filed May 12, 2004, is directed to mini chaperones and methods of use thereof for the treatment of cancer and other disorders. Also disclosed are tools to facilitate screening therapeutic agents that have selective binding affinity for GRP94. However, U.S. Pat. No. 7,589,174 is silent regarding the role of the Pre-N domain in glycosylation of minor glycosylation sites in Grp94.
U.S. Pat. No. 8,404,805 to Nicchitta et al., titled “CHARACTERIZATION OF GRP94-LIGAND INTERACTIONS AND PURIFICATION, SCREENING, AND THERAPEUTIC METHODS RELATING THERETO,” filed Oct. 5, 2009 is directed to characterization of interactions between ligands and Hsp90 proteins, including GRP94, wherein ligand binding to the N-terminal nucleotide binding domain of GRP94 elicits a conformational change that converts the GRP94 from an inactive to an active conformation, and wherein the chaperone and peptide-binding activities of the GRP94 are markedly stimulated. Also disclosed are purification, screening, and therapeutic methods pertaining to the biological activity of GRP94, and in some instances HSP90, based upon the characterization of ligand interactions of Hsp90 peptide-binding proteins, including GRP94. However, U.S. Pat. No. 8,404,805 fails to appreciate that the Pre-N domain regulates glycosylation of minor glycosylation sites in Grp94.
Accordingly, there remains a need in the art to control glycosylation of proteins for use as therapeutics, in screening assays, as research tools, and in the production of antibodies.
A challenge in the field of recombinant protein production is achieving substantially the same glycosylation pattern as the native protein, so that the recombinant protein exhibits substantially the same structure, function, activity, etc. The ability to control glycosylation of biologics, drug targets, antigens, and other proteins of interest would constitute an advancement in the art. Therefore, the inventive subject matter includes recombinant Grp94 proteins, mutants, and fragments thereof capable of regulating glycosylation of glycosylation sites downstream of the Grp94 Pre-N domain. The inventive subject matter also includes fusion proteins comprising the recombinant Grp94, mutants, and fragments thereof, therapeutic proteins, drug screening targets, antibodies, and other proteins of interest and methods of making and using the same.
In one embodiment, a method for glycosylation of Grp94 chaperone proteins expressed in mammalian cells is presented. For instance, the method comprises altering a Pre-N domain of the Grp94 chaperone protein, wherein the alteration of the Pre-N domain of the Grp94 chaperone protein leads to glycosylation at available glycosylation sites downstream of the Pre-N domain of the Grp94 chaperone protein, when expressed in mammalian cells.
In another embodiment, presented herein is a method for full glycosylation of any protein expressed as a fusion protein at a C-terminus of a Grp94 chaperone protein when the protein is expressed in mammalian cells. The method comprises altering a Pre-N domain of the Grp94 chaperone protein.
In a further embodiment, presented herein is a method for full glycosylation of any protein expressed as a fusion protein at a C-terminus of a Pre-N domain of a Grp94 chaperone protein, when the protein is expressed in mammalian cells. The method comprises altering a Pre-N domain of the Grp94 chaperone protein.
The alterations may include one or more of the following: deleting the Pre-N domain of the Grp94 chaperone protein; deleting residues 22-47 of the Pre-N domain; deleting one or more amino acids of the Pre-N domain of the Grp94 chaperone protein; substituting residues of the Pre-N domain with a different amino acid; or inserting one or more amino acids into the Pre-N domain, at any position.
In one embodiment, a recombinant fusion protein includes Grp94 and a glycosylated protein fused to the C-terminus of Grp94. The Grp94 may be wild type Grp94 or a recombinant Grp94. The glycosylated protein is glycosylated at one or more glycosylation sites as a result of being expressed downstream from the Grp94. Exemplary glycosylated proteins may be glycosylated at one, two, three, four, five or more glycosylation sites following expression of the recombinant fusion protein in a host cell. In some embodiments, the glycosylated protein in the recombinant fusion protein is glycosylated at all potential glycosylation sites, i.e., fully glycosylated.
In an embodiment that includes the recombinant Grp94, suitable recombinant Grp94 include substitution of five or more residues or deletion of seven or more residues from a Pre-N domain of the recombinant Grp94. For example, the recombinant Grp94 may include deletion of residues 22 to 32 from the Pre-N domain (e.g., SEQ ID NO. 2), deletion of residues 22 to 47 from the Pre-N domain (e.g., SEQ ID NO. 3, or deletion of the Pre-N domain (e.g., SEQ ID NO. 4).
The recombinant Grp94 may also include substitution of five or more residues of the Pre-N domain with alanine. Residues 33 to 37 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 5). Residues 38 to 42 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 6). Residues 33 to 37 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 7). Residues 48 to 52 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 8).
A further embodiment includes methods of producing any of the recombinant fusion proteins described herein. The method includes a step of culturing a host cell including a vector, which comprises a nucleic acid encoding the recombinant fusion protein under conditions permitting the production of the recombinant fusion protein. Exemplary vectors include (i) SEQ ID NO. 35, SEQ ID NO. 36, or SEQ ID NO. 34, which encode Grp94 and in which several residues are deleted from the Pre-N domain, and (ii) the nucleic acid sequence encoding the protein fused to the Grp94. Suitable vectors also include (i) SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, or SEQ ID NO. 51, which encode Grp94 and in which several residues are substituted with alanine, and (ii) the nucleic acid sequence encoding the protein fused to the Grp94. The recombinant fusion protein may also be recovered.
In another embodiment, a method of glycosylating a protein includes the steps of expressing any of the recombinant fusion proteins described herein in a host cell. When the recombinant fusion protein includes a cleavable linker, the method may include a step of cleaving the protein from the wild type or recombinant Grp94. The recombinant fusion protein may also include a tag, such as a histidine tag, a flag tag, or a biotin tag that facilitates isolation of the recombinant fusion protein or the tagged portion thereof. In some embodiment(s), the protein is glycosylated at a glycosylation sequon, such as Asn-X-Ser or Asn-X-Thr (X is any amino acid except proline). Suitable host cells may be mammalian, including human embryonic kidney line (e.g., HEK293).
In further embodiments, the glycosylated protein produced by the methods described herein is a drug target and may be used as substrates for drug screens. In another embodiment, the glycosylated protein is a therapeutic protein that may be formulated into a pharmaceutical composition and optionally included in a kit with reagents, supplies, and/or instructions for use. In another embodiment, the glycosylated protein is an antigen that can be used to produce antibodies for use in assays, protein purification, or as therapeutic antibodies.
The above embodiments are intended to be exemplary only, wherein other suitable embodiments will be apparent to those of sufficient skill. These other embodiments are within the scope of the disclosed subject matter. Furthermore, a number of references are cited throughout this application, each of which are herein incorporated by reference in their entirety.
So that the manner in which the features of the disclosure can be understood, a detailed description may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments and are therefore not to be considered limiting of its scope, for the scope of the disclosed subject matter encompasses other embodiments as well. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments. In the drawings, like numerals are used to indicate like parts throughout the various views.
Corresponding reference characters indicate corresponding parts throughout several views. The examples set out herein illustrate several embodiments, but should not be construed as limiting in scope in any manner.
The present disclosure relates to using the Grp94 Pre-N Domain to regulate N-linked glycosylation in order to preserve chaperone function. Many proteins in mammalian cells that are expressed in the endoplasmic reticulum are co- or post-translationally modified by N-linked glycosylation at glycosylation sequons (N-X-S/T, where X is any amino acid but proline) in the amino acid sequence of the protein.
Protein sequences may contain many N-linked glycosylation sequons, but these sites may not always be modified when the protein is expressed in mammalian cells. Many sites are silent or are used only under certain conditions.
Advantageously, the present technique may be used for scientific, diagnostic, or therapeutic applications to produce a protein in mammalian cells that is fully glycosylated at one or more of the glycosylation sites. The present method directs the cellular machinery to attach glycosylation groups at all available sequons, whether these sequons occur naturally or are introduced into the DNA coding for the protein by mutagenesis.
Grp94 is a highly abundant endoplasmic reticulum (ER) lumenal chaperone that is essential for the maturation of a restricted, yet important subset of proteins in the secretory pathway. The mechanisms by which Grp94 is regulated in the rapidly fluctuating folding environment of the ER remain largely unexplored. Here, the inventors report that N-linked glycosylation inactivates client maturation function. Applicant has found that this process is intrinsically regulated by the Pre-N domain, which serves a protective role by inhibiting glycosylation at functionally deleterious sites. When the Pre-N domain is truncated or mutated, normal mono-glycosylation is fundamentally disrupted, leading to hyper-glycosylation and loss-of-function. Applicant has further found that hyper-glycosylation alters the biochemical characteristics and ATPase activity of Grp94 depending on the availability of certain sites, suggesting that differential glycosylation may tune Grp94 chaperone activity and promote new biological functions. In sum, these findings suggest a molecular mechanism by which the Pre-N domain controls N-linked glycosylation and provide new insight into the regulatory function of this modification for Grp94.
Most newly synthesized proteins destined for the cell surface or secretion must transit through the endoplasmic reticulum (ER) for folding and post-translational processing (Schwarz and Blower, 2016). Grp94 is the ER resident member of the hsp90 family of chaperones and it is specifically required for the maturation of various membrane and secreted proteins, such as GARP, pro-insulin, insulin-like growth factors (IGFs), platelet glycoprotein Ib-IX-V complex, and most members of the LDLR, toll-like receptor (TLR), and integrin families (Zhang et al., 2015) (Ansa-Addo et al., 2016) (Ghiasi et al., 2019) (Randow and Seed, 2001) (Weekes et al., 2012) (Yang et al., 2007). Grp94's control over the fate of these diverse proteins makes it an emerging therapeutic target for number of diseases including, but not limited to type 2 diabetes, familial hypercholesterolemia, and sepsis (Ghiasi et al., 2019) (Poirier et al., 2015) (Li et al., 2019), as well as cancers such as HER2-positive breast cancer and multiple myeloma (Patel et al., 2013) (Liu et al., 2013) (Ansa-Addo et al., 2016). A recent proteomics study identified a much larger set of Grp94-associated proteins (Hong et al., 2017), deepening Applicant's understanding of the cellular importance of this paralog.
Like its hsp90 family members, Grp94 is a homodimeric ATP-driven machine containing three (3) major domains: the N-terminal (NTD), Middle (MD), and C-terminal (CTD) domains (Dollins et al., 2007a; Huck et al., 2017a). The NTD is the site of ATP binding, the MD is involved in ATP hydrolysis, and the CTD is the site of the high affinity homo-dimerization interface (Prodromou, 2016). In addition to the three core domains, hsp90s possess divergent regions of variable length, including a Pre-N domain (Pre-N) that precedes the NTD, a Charged Linker (CL) domain between the NTD and MD, and a C-terminal extension (CX) from the CTD (Maharaj et al., 2016). The hsp90 chaperone cycle involves a series of large conformational shifts between open and closed dimer states that are driven by the binding of ATP (Krukenberg et al., 2011). Recent structural and biophysical studies have begun to clarify how Hsp90 engages and matures its clients (Kirschke et al., 2014) (Karagoz et al., 2014) (Verba et al., 2016) (Dahiya et al., 2019) (Boysen et al., 2019). However, the mechanisms by which Grp94 utilizes ATP to activate its own diverse group of clients remain poorly understood.
ER proteostasis is achieved by matching chaperone availability with protein folding load (Gardner et al., 2013). Despite being required for only a limited subset of the proteins that transit the ER, Grp94 is the most abundant ER protein (Itzhak et al., 2016). This apparent contradiction raises the question as to how Grp94 activity is regulated within a dynamic folding environment to ensure efficient folding under non-stress conditions (Braakman and Hebert, 2013) (Schwarz and Blower, 2016). In addition, ER stress conditions, such as the unfolded protein response (UPR), oxidative imbalance, glucose deprivation, calcium perturbation, hypoxia, and glycosylation blockade, as well as certain cancers further induce expression of Grp94 and other ER chaperones in order to meet the increased folding demands and assist in proteasomal degradation of misfolded proteins (Zheng et al., 2008). BiP, another highly abundant ER chaperone, is the central mediator of protein folding and stress response in the ER. It is now appreciated that BiP possesses a multitude of regulatory mechanisms such as oligomerization, AMPylation, and co-factor modulation to regulate activity in response to changes in client protein load (Preissler et al., 2015a) (Preissler et al., 2015b) (Marcinowski et al., 2011). Likewise in the cytosol, Hsp90 possesses several layers of regulation including various post-translational modifications and dedicated co-chaperones that finely tune ATP hydrolysis activity and client recruitment (Schopf et al., 2017).
In contrast, the regulatory roles of protein modifications and co-chaperones for Grp94 function have long been obscure. N-linked glycosylation is an essential co-translational and, in some cases, post-translational modification of proteins that transit the ER due to its critical roles in protein folding, secretion, and protein-protein interactions (Helenius and Aebi, 2004). N-linked glycosylation is carried out by the oligosaccharyltransferase (OST) complex on asparagine (Asn) residues in the consensus sequon Asn-X-Ser/Thr, where X can be any amino acid except proline (Schwarz and Aebi, 2011). Here, “sequon” refers to the N-linked glycosylation motif (Asn-X-Ser/Thr), whereas “site” specifically refers to the modified Asn residue. Grp94 contains six sequons with acceptor sites at Asn62 in the Pre-N domain, Asn107 and Asn217 in the NTD, and Asn445, Asn481, and Asn502 in the Middle domain (MD). Asn217 is the major site of glycosylation and is nearly constitutively modified under normal conditions (Qu et al., 1994; Wearsch and Nicchitta, 1996). The remaining glycosylation sites have been collectively referred to as minor, cryptic, or silent, to reflect their atypical usage (Dersh et al., 2014b)(Cherepanova et al., 2019). Multi-glycosylated forms of Grp94 have been observed (Qu et al., 1994) (Feldweg and Srivastava, 1995) (Cala, 2000) (Yang et al., 2007) (Dersh et al., 2014b) (Seidler et al., 2014), but the biological importance of these species is almost completely unknown. Furthermore, unlike its cytosolic counterpart, Grp94 does not possess a network of co-chaperones to regulate ATP hydrolysis and client binding. Paralogs of cytosolic co-chaperones do not exist in the ER and the only bona fide co-chaperones identified to date include CNPY proteins, which appear to be client specific (Liu et al., 2010) (Rosenbaum et al., 2014).
It has been recently discovered that the Pre-N domain is required for the maturation and cell surface localization of Grp94 client integrins and TLRs (Huck et al., 2017b). The Pre-N domain is a conserved extension preceding the Grp94 NTD, yet it varies substantially in length and sequence among hsp90s, with the Grp94 Pre-N domain (52 amino acids) dwarfing the Pre-N regions of the other paralogs (Trap 1: 24 aa; Hsp90a: 16 aa; Hsp90(3: 11 aa). Given that the Pre-N domain is both intrinsically suppressive of ATP hydrolysis and that it is essential for client maturation, the inventors postulated previously that this unique region might replace some of the mechanistic functions of a regulatory co-chaperone in the ER by, for example, recognizing and recruiting clients or stabilizing Grp94:client complexes. Here, the inventors set out to uncover the essential role of the Pre-N domain in Grp94 chaperone function. Surprisingly and unexpectedly, the inventors found that the Pre-N domain controls the N-linked glycosylation status of Grp94. Truncations and mutations of the Pre-N domain lead to high occupancy glycosylation that render the chaperone non-functional; an effect that can be reversed by mutating deleterious glycosylation sites to prevent their modification. Applicant has further found that hyper-glycosylation alters the biochemical properties and the ATPase activity of Grp94 depending on the availability of certain sites, suggesting that differential N-linked glycosylation may serve to tune Grp94 activity in the cell and promote new biological functions. Taken together, Applicant's results may explain the evolutionary importance of the Pre-N domain and provide clues into the regulatory roles of N-linked glycosylation for Grp94.
In one embodiment, a recombinant Grp94 protein is derived from human Grp94, monkey Grp94, or canine Grp94. In another embodiment, a recombinant fusion protein includes Grp94 and a glycosylated protein fused to the C-terminus of Grp94. The Grp94 in the fusion protein be wild type Grp94 or a recombinant Grp94 as described herein. Non-limiting examples of Grp94 chaperones that may be employed in the inventive compositions and methods include those listed in Table 1 (SEQ ID NOS. 1-32), encoded by the corresponding DNA sequences (SEQ ID NOS. 33-64). Protein and DNA sequences having about 95%, about 96%, about 97%, about 98%, or about 99% sequence homology are also contemplated.
In an embodiment that includes the recombinant Grp94, suitable recombinant Grp94 include substitution of five or more residues or deletion of seven or more residues from a Pre-N domain of the recombinant Grp94. In some embodiments, recombinant Grp94 includes deletion of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 amino acid residues from the Pre-N region. In some embodiments, recombinant Grp94 may include deletion of about 13% to 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 70%, about 70% to about 90%, or about 90% to about 95% of the residues from the Pre-N domain. It should be appreciated that such deletions may be random or sequential. Deletions may begin with amino acid residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or any amino acid residue downstream therefrom, provided there are sufficient amino acid residues to be deleted from the Pre-N domain. For example, the recombinant Grp94 may include deletion of residues 22 to 32 from the Pre-N domain (e.g., SEQ ID NO. 2), deletion of residues 22 to 47 from the Pre-N domain (e.g., SEQ ID NO. 3), or deletion of the Pre-N domain (e.g., SEQ ID NO. 4). In some embodiments, the recombinant Grp94 has about 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4.
The recombinant Grp94 may also include substitution of five or more residues of the Pre-N domain. In some embodiments, recombinant Grp94 may include substitution of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 amino acid residues in the Pre-N region. In some embodiments, recombinant Grp94 may include substitution of about 13% to 15%, about 15% to about 25%, about 25% to about 50%, about 50% to about 70%, about 70% to about 90%, or about 90% to about 95% of the residues from the Pre-N domain. It should be appreciated that such substitutions may be random or sequential. Deletions may begin with amino acid residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or any amino acid residue downstream therefrom, provided there are sufficient amino acid residues to be substituted from the Pre-N domain. In one embodiment, amino acid residues in the Pre-N region of Grp94 are substituted with alanine. In another embodiment, amino acid residues in the Pre-N region of Grp94 are substituted with alanine, glycine, valine, leucine, and/or isoleucine. Residues 33 to 37 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 5). Residues 38 to 42 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 6). Residues 33 to 37 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 7). Residues 48 to 52 of the Pre-N domain may be substituted with alanine (e.g., SEQ ID NO. 8).). In some embodiments, the recombinant Grp94 has about 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8. In some embodiments, the Pre-N domain of recombinant Grp94 includes a combination of deletion and substitution of residues.
Suitable glycosylated proteins (also referred to as glycoproteins) are listed in Table 2. In one embodiment, the glycosylated protein comprises a fragment of one of the glycoproteins listed in Table 2. In another embodiment, the glycosylated protein comprises a mutant of one of the glycoproteins listed in Table 2. In a further embodiment, the glycosylated protein is a fusion protein comprising a fragment of one of the glycoproteins listed in Table 2 and another protein. The glycosylated protein may be fused to Grp94 directly or indirectly via a linker. Suitable linkers include Tobacco Etch Virus protease cleavage sites (Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)) and those described by Xiaoying Chen et al. in Design of an in vivo cleavable disulfide linker in recombinant fusion proteins, 49 B
A further embodiment includes methods of producing any of the recombinant fusion proteins described herein. The method includes a step of culturing a host cell (e.g., HEK293), including a vector which comprises a nucleic acid encoding the recombinant fusion protein under conditions permitting the production of the recombinant fusion protein. Exemplary vectors include (i) SEQ ID NO. 35, SEQ ID NO. 36, or SEQ ID NO. 34, which encode Grp94 in which several residues are deleted from the Pre-N domain, and (ii) the nucleic acid sequence encoding the protein fused to the Grp94. Suitable vectors also include (i) SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, or SEQ ID NO. 51, which encode Grp94 in which several residues are substituted with alanine, and (ii) the nucleic acid sequence encoding the protein fused to the Grp94. It should be appreciated that vectors may include DNA sequences having about 95%, about 96%, about 97%, about 98%, or about 99% sequence homology with SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 34, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, or SEQ ID NO. 51. The recombinant fusion protein may also be recovered.
In another embodiment, a method of glycosylating a protein includes the steps of expressing any of the recombinant fusion proteins described herein in a host cell. When the recombinant fusion protein includes a cleavable linker (e.g., a protease cleavable linker), the method may include a step of cleaving the protein from the wild type or recombinant Grp94. The recombinant fusion protein may also include a tag, such as a histidine tag, a flag tag, or a biotin tag that facilitates isolation of the recombinant fusion protein or the tagged portion thereof. In some embodiments, the protein is glycosylated at a glycosylation sequon, such as Asn-X-Ser or Asn-X-Thr (X is any amino acid except proline). In a further embodiment, the recombinant fusion protein is coexpressed with oligosaccharyltransferase (OST) complex.
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a Grp94 protein (or a portion thereof), or a Grp94 fusion protein. Grp94 proteins, functional fragments thereof, and fusion proteins can be produced from wild type Grp94 sequences found in animals (e.g., human, canine, mouse, rat, cow, sheep, goat and birds), insect cells, plants, yeast or bacteria, as known in the art using the methods described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., Grp94 proteins, Grp94 protein fragments, Grp94 fusion proteins, and the like).
The recombinant expression vectors of the invention can be designed for expression of Grp94 proteins and fusions in prokaryotic or eukaryotic cells. For example, Grp94 proteins and fusions can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three (3) purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Purified glycosylated proteins or Grp94 fusion proteins can be utilized in protein activity assays, (e.g., direct assays or competitive assays described in detail herein), or to generate antibodies specific for glycosylated proteins or Grp94 fusion proteins, for example.
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166).
Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
Suitable mammalian cells that can be used to obtain Grp94 proteins, fusion proteins, or functional fragments include primary cell cultures derived from a mammal at any stage of development or maturity. Mammalian cells also include cells of mammalian origin that have been transformed to divide for an unlimited number of generation, such as human embryonic kidney line (e.g., HEK293), human fibrosarcoma cell line (e.g., HT1080), human cervical carcinoma cells (HeLa), human lung cells (W138), human liver cells (Hep G2), human retinoblasts, BALB/c mouse myeloma line, COS-7, baby hamster kidney cells (e.g., BHK), Chinese hamster ovary cells (e.g., CHO+/−DHFR), mouse Sertoli cells (TM4), rat liver cells (BRL 3A), mouse mammary tumor (e.g., MMT-060562), TRI cells; MRC 5 cells, FS4 cells, monkey kidney cells (e.g., CV1, VERO-76), and canine kidney cells (e.g., MDCK). Different host cells can be chosen to ensure its capacity to modify and process Grp94 proteins, fusion proteins, or functional fragments thereof.
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to Grp94 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types; for instance, viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the terms as used herein.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an Grp94 protein or fusion protein, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
Suitable host cells may be mammalian. Exemplary host cells include: human embryonic kidney line (e.g., HEK293), human fibrosarcoma cell line (e.g., HT1080), human cervical carcinoma cells (HeLa), human lung cells (W138), human liver cells (Hep G2), human retinoblasts, BALB/c mouse myeloma line, COS-7, baby hamster kidney cells (e.g., BHK), Chinese hamster ovary cells (e.g., CHO+/−DHFR), mouse Sertoli cells (TM4), rat liver cells (BRL 3A), mouse mammary tumor (e.g., MMT-060562), TRI cells; MRC 5 cells, FS4 cells, monkey kidney cells (e.g., CV1, VERO-76), and canine kidney cells (e.g., MDCK).
In further embodiments, the glycosylated protein produced by the methods described herein is a drug target and may be used as substrates for drug screens. In another embodiment, the glycosylated protein is a therapeutic protein that may be formulated as a pharmaceutical composition with a pharmaceutically acceptable excipient and optionally included in a kit with reagents, supplies, and/or instructions for use. In another embodiment, the glycosylated protein is an antigen that can be used to produce antibodies for use in assays, protein purification, or as therapeutic antibodies.
In some embodiments, recombinant Grp94 proteins (including, but not limited to chimeric proteins) disclosed herein and/or antibodies directed to such proteins may be developed using assays described herein. In some embodiments, recombinant proteins (including, but not limited to chimeric proteins) disclosed herein and/or antibodies directed to such proteins may be used in assays to develop other recombinant proteins and/or antibodies of the present invention.
Binding Assays. In some embodiments, the present invention provides binding assays. As used herein, the term “binding assay” refers to an assay used to assess the ability of two or more factors to associate. Such assays may assess the ability of a desired antigen to bind a desired antibody and then use one or more detection methods to detect binding. Binding assays of the invention may include, but are not limited to surface Plasmon resonance-based assays, ELISAs and FACS-based assays. Binding assays of the invention may comprise the use of one or more recombinant proteins described herein, including, but not limited to any glycosylated proteins, any chimeric proteins, any cofactors and any modules, combinations or fragments thereof.
Cell-Based Assays. In some embodiments, the present invention provides cell-based assays. As used herein, the term “cell-based assay” refers to an assay comprising at least one aspect that involves the use of a living cell or cell culture. In some embodiments, these may be useful for assessing the modulation of protein activity based on the degree of glycosylation. Cell-based assays of the present invention may comprise expression cells and/or responsive cells. Expression cells, as referred to herein, are cells that express one or more factors being analyzed in a particular assay. Such expression may be natural or may be the result of transfection and/or transduction of a foreign gene. In some embodiments, expression of one or more factors by expression cells may be enhanced or suppressed by the addition of one or more exogenous factors. In some embodiments, expression cells may comprise cell lines (e.g. HEK293 cells, CHO cells, TMLC cells, 293T/17 cells, Hs68 cells, CCD1112sk cells, HFF-1 cells, Keloid fibroblasts or Sw-480 cells.) In some embodiments, cell lines comprising expression cells may express one or more recombinant proteins of the present invention (e.g. naturally and/or through transfection, stable transfection, and/or transduction).
In one embodiment, a method for manufacturing an antibody, or an antigen-binding portion thereof, includes the steps of: (i) providing a glycosylated antigen produced by the methods described herein; and (ii) selecting a pool of antibodies, or antigen-binding portion thereof, for the ability to bind the glycosylated antigen of step (i). In some embodiments, the antibody, or antigen-binding portion thereof is a human or humanized antibody or antigen-binding portion thereof. In some embodiments, the method further includes a step of immunizing a host animal with the antigen of step (i). Optionally, the method may include collecting lymphocytes from the host animal that bind the antigen of step (i).
In one embodiment, step (ii) includes screening a library, e.g., an antibody display library. In another embodiment, step (ii) includes screening an antibody fragment display library, e.g. a library of Fab fragments, and/or single-chain variable fragments (scFvs). The antibody, or antigen-binding portion thereof, may be an IgA, IgD, IgE, IgG or IgM antibody.
In one embodiment, a method for manufacturing a pharmaceutical composition comprising an antibody, or an antigen-binding portion thereof, includes the steps of: (i) providing a glycosylated antigen produced by the methods described herein; (ii) selecting a pool of antibodies, or antigen-binding portion thereof, for the ability to bind the glycosylated antigen of step (i); (iii) selecting from the pool of antibodies, or antigen-binding portions thereof, an antibody, or an antigen-binding portion thereof that exhibits a desired effect; and, (iv) formulating the antibody or antigen-binding portion thereof from step (iii) into a pharmaceutical composition, wherein the antibody, or antigen-binding portion thereof is a human or humanized antibody or antigen-binding portion thereof.
Experimental procedures, including cloning and construct generation, are now discussed with respect to a specific working example. All constructs used in these studies were derived from canine Grp94 (UniProt P41148), which is −98% identical to human Grp94. For mammalian expression, full-length and Pre-N truncated Grp94 genes were generated by PCR and inserted into the pTT5 vector between NotI and BamHI restriction sites. For bacterial expression, Grp94 was cloned into the NdeI and BamHI sites of the pET15b vector. All constructs were confirmed by sequencing.
Protein expression and purification in mammalian cells were carried out as follows: Mammalian cells were cultured in Freestyle293 media (Thermo Fisher) in a 37° C. incubator with 5% humidified CO2 and shaken at 140 rpm. Cells were transfected using a 4 to 1 ratio of branched PEI (Sigma) to vector and cultured for 24-72 hours as indicated before harvesting.
For purification of Grp94 for biochemical and ATPase analyses, cultures were transfected with 0.7-0.8 μg DNA per million cells and incubated for 72 hours. Cells were harvested by centrifugation at 500×g for 3 minutes, the culture supernatant was removed, cells were washed in ice-cold PBS, and then lysed by incubating in buffer consisting of 50 mM Tris-HCl (pH 8.0), 350 mM NaCl, 20 mM imidazole, and 0.5% CHAPS on ice for 30 minutes with periodic mixing. Lysis debris was cleared by centrifugation at 10,000×g for 5 minutes at 4° C. Ni-NTA agarose (Qiagen) was then added in batch to the lysate and allowed to capture His-tagged proteins overnight with nutation at 4° C. After washing with lysis buffer containing decreasing amounts of detergent, protein was stepwise eluted from the resin with buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 350 mM imidazole, and 1 mM β-me. Eluted proteins were then injected onto a Superdex 200 16/60 (GE Healthcare Life Sciences) size-exclusion column equilibrated in 40 mM HEPES-KOH (pH 7.6), 150 mM KCl, and 5 mM MgCl2. Peak fractions were pooled and concentrated before flash freezing in liquid nitrogen.
Protein expression and purification in E. coli was carried out as follows. Proteins were expressed and purified as previously described (Dollins et al., 2007b) (Huck et al., 2017b). Briefly, constructs were expressed in BL21-Star (DE3) (Invitrogen) as N-terminal hexa-histidine fusion proteins. Cultures were typically grown at 37° C. and induced at mid-log with IPTG to a final concentration of 0.5-1.0 mM. The protein purification for all constructs consisted of Ni-affinity, Q-Sepharose anion exchange, and gel filtration purification steps. Protein fractions were concentrated between 10-30 mg/ml, aliquoted, and flash-frozen in liquid nitrogen.
For western blot analysis of mammalian cell lysates, cells were harvested by centrifugation at 500×g for 3 minutes following 24 or 48 hours of expression. The culture supernatant was removed, cells were washed in ice-cold PBS, and then lysed by incubating in buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, and 0.1% SDS on ice for 30 minutes with periodic mixing. Lysis debris was cleared by centrifugation at 10,000×g for 15 minutes at 4° C. Prior to western blot, samples were quantified using the Pierce BCA protein assay kit. In general, samples were mixed with 5× Laemmli buffer and run on 7.5% or 4-15% SDS-PAGE gels as indicated. Gels were run at 100 V for 2-2.5 hours using Tris-glycine SDS running buffer and transferred onto Bio-Rad Immun-Blot PVDF membranes using a BioRad Semi-Dry apparatus at 12 V for 70 minutes in transfer buffer. Membranes were blocked for 1 hour using 5% w/v non-fat milk in TBS-T (Tris-buffered saline with 1% Tween-20) and then incubated with primary antibodies diluted in 2% milk in TBS-T overnight at 4° C. or for 2 hours at room temperature. All primary antibodies were used at a final dilution of 1:1,000 or 1:2000. Membranes were then washed with 1×TBS-T and incubated with HRP-conjugated secondary antibodies (from) diluted 1:10,000 in 2% milk in TBS-T for 1 hour at room temperature. Membranes were washed in 1×TBS-T and chemiluminescent substrate (Thermo-Fisher SuperSignal West Pico) was added. Signal was captured using a Bio-Rad Chemi-Doc imaging system. Densitometry was performed using ImageJ.
Purified Grp94 samples were partially de-glycosylated with Endo-H (New England Biolabs) prior to mass spectrometry analysis. 150 μg of protein were denatured by adding 1× denaturing buffer and heating to 99° C. for 8 minutes followed by cooling on ice. De-glycosylation was achieved by adding 1× Glycobuffer-3 and 5 μL of Endo-H (2500 units) and incubating at 37° C. for 2 hours. De-glycosylation was confirmed by SDS-PAGE. Endo-H treated Grp94 was then precipitated using TCA to remove detergents, salts, and sugars. The mixture was incubated on ice for 30 minutes and then spun at 14,000×g for 5 minutes to pellet the precipitant. The supernatant was removed and the pellet was washed twice with 200 μL of ice-cold acetone. The pellet was dried by heating the tube to 95° C. using a heat block for 1-2 minutes. The dry pellet was re-suspended in 20 μL of 8 M urea (in 0.1 M Tris-HCl pH 8.5) and subsequently diluted to 2 M urea. Tris(2-carboxyethyl)phosphine (TCEP) was added to 5 mM final and the samples were heated to 95° C. for 10 minutes to reduce disulfide bonds. Freshly prepared iodoacetamide was then added for a final concentration of 10 mM and incubated at room temperature for 30 minutes to alkylate free cysteines. The samples were then buffer exchanged into 100 mM NH4HCO3 using a 7-kDa MWCO Zeba desalting column (Thermo Scientific) according to the manufacturer's protocol. Finally, the samples were proteolyzed by adding 2.25 μg of Trypsin Gold (Promega) or 2.3 μg of Endoproteinase LysC (New England Biolabs) and incubating at 37° C. for ˜40 hours with shaking. Proteolysis was confirmed by SDS-PAGE.
Mass spectrometry (MS) analysis was performed using an Orbitrap-XL spectrometer (Thermo) equipped with a nano-LC column (PepMap C18 2 μm; 75 μm x150 mm, Thermo) and ESI (electrospray ionization) source. In the Orbitrap-XL runs, the mobile phases were, A: water and B: acetonitrile (CH3CN), both containing 0.1% (v/v) formic acid. Data were acquired over 70 min using the following gradient: linear at a flow rate of 300 nL/min: (i) increase from 0% to 10% B over 5 min; (ii) 10% to 45% B over 60 min; (iii) 45% to 100% B over 10 min; and, finally, (iv) isocratic elution at 100% B over 10 min. MS data were collected over 600-2000 m/z in positive ion mode. MS1 data were acquired using the Orbitrap detector (60,000 resolution), and MS/MS in CID mode (ion trap with 30% collision energy). Data analysis was performed using the glycoproteomics toolbox GlycoPAT (Liu et al., 2017), and the Uniprot protein sequence P41148 containing 6 N-X-S/T sites at N62, N107, N217, N445, N481 and N502. Search was performed for a single HexNAc in the case of the EndoH treated samples, and using an N-glycan library with 227 glycans for carbohydrate epitope mapping. Following manual validation of all hits with GlycoPAT ensemble score (ES) >0.4, peak area under the curve was quantified using Xcalibur Quan Browser 3.0 (Thermo).
Cell lines and antibodies for client maturation assay: Grp94-null E4.126 pre-B cells were a gift from Dr. B. Seed (Harvard University, Cambridge, Mass.). Pre-B cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 10% FBS (Atlas Biologicals), and 0.055 mM β-me (Life Technologies). Phoenix Eco cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS and same concentration of penicillin and streptomycin as above. E4.126 and Phoenix Eco cells were cultured at 37° C. with 5% CO2. Grp94 antibody 9G10 was purchased from Enzo Life Sciences and detected both endogenous and overexpressed proteins. The anti-FLAG M2 antibody was purchased from Sigma. Biotin-conjugated anti-mouse CD11a (Clone: M174), CD49d (Clone: R1-2), and TLR2 (Clone: 6C2) antibodies were purchased from Affymetrix and were used to detect endogenous proteins.
Retrovirus production and transduction were carried out as follows. Ecotropic retrovectors (MigR1) containing Grp94 and EGFP were transfected into Phoenix Eco cells using Lipofectamine 2000 (Invitrogen). Two days later, virus-containing medium was collected and added to 2×105 E4.126 cells, along with hexadimethrine bromide (Sigma-Aldrich). Spin infection was achieved by centrifugation at 1900×g for 1.5 hours at 32° C. to facilitate viral transduction.
Flow cytometry analysis was carried out as follows. After retroviral transduction, cell surface expression of the Grp94 client (CD1 1a, CD49d, or TLR2) was evaluated by staining with a client-specific biotin conjugated primary antibody at 4° C. for 30 minutes. Detection was achieved by secondary antibody staining for 30 minutes at 4° C. with a streptavidin-conjugated Allophycocyanin (APC) fluorescent antibody that binds to the biotin-conjugated primary antibody. A biotin-conjugated isotype antibody was used as a negative control to assess background levels. For intracellular staining, which was used to assess the overexpression level of FLAG-tagged Grp94 and the chimeras, cells were fixed in 4% paraformaldehyde at room temperature, and then permeabilized with ice-cold methanol at −20° C. to allow antibody entry. Normal goat serum was used for blocking prior to anti-FLAG antibody or isotope control antibody staining. Antibody staining procedures were similar to surface staining, except all steps were done at room temperature. Antibody or isotype stained cells were acquired on a FACSCalibur (BD Biosciences) and analyzed using the FlowJo software. Using EGFP expression as a positive indicator for retroviral infection, the EGFP-positive cells were analyzed for both surface expression of matured Grp94 clients as well as for the intracellular expression of Grp94.
Size-exclusion chromatography experiments were carried out as follows. Purified Grp94 proteins were adjusted to 5.5 μM in buffer consisting of 40 mM HEPES-KOH (pH 7.4), 150 mM KCl, and 5 mM MgCl2 at a final volume of 1 mL before injection. Each sample was injected onto a Superdex 200 16/60 (GE Healthcare Life Sciences) size-exclusion column equilibrated in 40 mM HEPES-KOH (pH 7.6), 150 mM KCl, and 5 mM MgCl2 at 4° C. at a flow rate of 1.0 mL/min. Absorbance at 280 nm was detected and plotted against the elution time. The void volume was measured using blue dextran (Sigma) as a marker.
Native gel electrophoresis: Samples for Native-PAGE analysis were prepared by pulling-down C-terminally His-tagged Grp94 from total lysates (prepared by detergent lysis in 0.5% CHAPS) using Ni-NTA resin, followed by elution in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 350 mM Imidazole, and 1 mM β-me. Eluted samples were quantitated by nanodrop. 2 μg of each sample was mixed with 2× Native-PAGE loading buffer and loaded on 4-15% Native gels (Biorad) and resolved for 120 minutes at 150V in Tris-Glycine running buffer under non-reducing conditions. The electrophoresis apparatus was kept cold on ice. The proteins were visualized by coomassie blue staining.
ATP Hydrolysis Assay: ATP hydrolysis rates were measured using the PiPer Phosphate assay kit (Thermo Fisher Scientific) in 96-well fluorescent assay plates (Corning). Assay components were prepared at the recommended stock concentrations in 1× reaction buffer (0.1 M Tris-HCl (pH 7.6)), flash frozen in liquid nitrogen, and stored at −20° C. A stock of −100 mM ATP was prepared in H2O, aliquoted, and frozen at −20° C. Phosphate standards from 0-160 μM were brought up in 2× ATPase buffer (80 mM HEPES-KOH (pH 7.4), 300 mM KCl, and 10 mM MgCl2). Purified proteins were exchanged into 40 mM HEPES-KOH (pH 7.4), 150 mM KCl, and 5 mM MgCl2 (1× ATPase buffer), concentrated to 50 and diluted prior to the experiment. The final concentration of protein in each well was 4.5 and the final ATP concentration varied from 0-800 μM. Experimental setup included a 50 μL:50 μL mixture of PiPer reagent (100 μM Amplex Red reagent containing 4 U/mL maltose phosphorylase, 0.4 mM maltose, 2 U/ml glucose oxidase, and 0.4 U/mL horseradish peroxidase) and the ATPase reaction (5 μL ATP stocks and 45 protein (10 μM stock for 4.5 μM final) or 45 μL 1× ATPase buffer for ATP only wells). Plates were incubated at 37° C. for 3 hours and the reactions were quenched on ice.
Fluorescence was measured at 544 nm/590 nm (excitation/emission) on a SpectraMax Gemini XS plate reader (Molecular Devices) with 30 readings per well. Data were corrected using the following equation: enzyme activity=full reaction (all components) −no enzyme control (ATP only background) −no substrate control (enzyme only)+no enzyme/no substrate control (buffer background). ATP hydrolysis experiments are averages of at least three independent measurements. The data were plotted using Prism and fit with the Michaelis-Menten equation.
Results of this working example are now discussed with respect to
Previous studies have shown that exogenous overexpression of Grp94 and pharmacological ER stressors induce the formation of a heavily glycosylated Grp94 species termed hyper-glycosylated Grp94 (hgGrp94) (Qu et al., 1994)(Cala, 2000)(Dersh et al., 2014b)(Seidler et al., 2014)(Cherepanova et al., 2019). The hgGrp94 species migrate more slowly than the normal, mono-glycosylated Grp94 (mgGrp94) by SDS-PAGE and western blot, giving rise to at least two distinct species that reflect the number of modified N-linked glycosylation sites. In particular, one study showed that large deletions made from the mature N-terminus of Grp94, including a region roughly corresponding to the domain boundaries of the Pre-N domain, forced the use of additional N-linked glycosylation sites beyond Asn217 (Qu et al., 1994). Based on these observations, we wanted to understand if the Pre-N domain plays a specific role in controlling the glycosylation pattern of Grp94. To do this, we expressed truncated Pre-N domain variants of Grp94 in mammalian cells and evaluated their electrophoretic mobility by western blot.
In agreement with previous studies, the inventors found that high overexpression of wild type Grp94 (Grp94WT) leads to the formation of a hyper-glycosylated species (
To better understand the impact of the Pre-N domain on hgGrp94 formation, the inventors transfected increasing amounts of FLAG-tagged Grp94WT and FLAG-tagged Grp94Δ22-32 plasmids (0.2-1.0 αg/106 cells) and monitored the presence of hgGrp94 compared to mgGrp94 by both α-FLAG and α-Grp94 (9G10) western blot. As hgGrp94 is of low abundance under endogenous conditions, the inventors predicted that hgGrp94 in a WT expression would increase as the folding demand for this protein rises. Indeed, a concentration-dependent increase in hgGrp94 was observed until saturation of overall expression appeared to have been reached at the 0.8 ag/106 cells transfection level (
The expression of different Pre-N-truncated constructs leads to hyper-glycosylation, but the individual usage and the percent occupancy of each site utilized in hgGrp94 is unknown. To better characterize hgGrp94, purified C-terminally FLAG-tagged Grp94Δ22-32 and Grp94ΔPreN recombinant proteins were purified by immunoprecipitation and subjected them to peptide glycosylation analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Prior to tryptic digestion, the samples were treated with Endo-H, which partially de-glycosylates high-mannose N-linked glycans, leaving behind an N-acetlyglucosamine (GlcNAc) moiety that is covalently linked to the asparagine residue. The resulting GlcNAc, if present, results in an increase in peptide mass compared to a non-glycosylated peptide, which can be used to identify which sites are modified and assess the relative frequencies of modified and unmodified peptides.
Peptide relative abundance quantifies % of peptide (both with and without GlcNAc) detected relative to all potentially glycosylated peptides. Here, label-free quantification of product area under the curve (AUC) is based on the ion current of the precursor mass in the LC-MS/MS chromatogram. All products are validated using MS/MS and LC retention time. n.d. indicates that the peptide could not be detected. Asn62 was likely detected in APre-N preparations due to contaminating endogenous protein. % Glycosylation=100*(AUC of peptide with GlcNAc)/(AUC of peptide with and without GlcNAc).
As summarized in Table 3, the inventors found high levels of glycosylation at all detectable sites (84% average, range 56-100%). (Glycosylation at Asn481 could not be determined, which is likely due to its proximity to multiple tryptic digest sites.) The hgGrp94 populations that result from the small (Δ22-32) and large (ΔPre-N) truncations were nearly identical, with each individual site being more glycosylated than not, suggesting that the glycosylation machinery does not skip specific sites during the process of hyper-glycosylation. Taken together, these results suggest that the Pre-N domain is involved in a specific mechanism to regulate the total amount of Grp94 glycosylation.
The correlation between hyper-glycosylation of Pre-N-truncated constructs and Applicant's previous discovery that the Pre-N domain is required for chaperone function (Huck et al., 2017a) raised the possibility that increased levels of glycosylation directly account for the loss of Grp94 client maturation activity. To determine if N-linked glycosylation at the minor sites is deleterious to chaperone function, the inventors employed an established client maturation assay using a Grp94-null murine pre-B cell line in which Grp94 client integrins (CD11a and CD49d) and TLRs (TLR2) are unable to reach the cell surface in the absence of exogenously expressed Grp94 (Randow and Seed, 2001).
As seen in
When the entire Pre-N domain is deleted and Asn62 is therefore removed, Grp94 retains one major (Asn217) and four minor (Asn107, Asn445, Asn481, and Asn502)N-glycosylation sites. To probe the functional impact of glycosylation at the minor sites, the inventors first mutated the three MD sites Asn445, Asn481, and Asn502 to alanines (MD-N3xA), which prevents their modification. Intriguingly, with the Pre-N domain deleted, the MD-N3xA construct partially restored the surface expression of integrins (14-15%) and TLR2 (10%) relative to WT, suggesting that glycosylation at one or more of these MD glycosylation sites are deleterious to chaperone function (
The positive effect of blocking glycosylation at Asn107 is not surprising. Modeling of a high-mannose N-glycan at Asn107 revealed that the addition of a sugar moiety at this residue would adversely affect ATP binding (
In addition, a large N-glycan tree would be incompatible with normal placement of the open lid conformation of the N-terminal domain and would sterically block the transition of Grp94 to the closed dimer conformation, which is necessary for ATP hydrolysis. Direct mutation of Asn107 to an alanine to block glycosylation is problematic from a mechanistic standpoint since Asn107 directly interacts with the phosphates of bound nucleotides. However, mutation of Ser109 to alanine within the Asn107-Ala108-Ser109 sequon also blocks glycosylation at Asn107 and places the mutation on the opposite face of the helix away from ATP, where it should not affect chaperone function. Mutation of all of the minor sequons does not negatively affect the function of the full-length protein. The right curves were gated on GFP+ cells. The number displayed on the histogram is the Mean Fluorescence Intensity (MFI) of these curves. Intracellular levels of the expressed C-terminally FLAG-tagged Grp94 were determined by staining with FLAG antibody. The left curves were produced by staining with an isotype control antibody (
Similar to the data with Grp94ΔPreN, the blocking mutations at all five minor sites, including Asn62 of the Pre-N domain, restore nearly complete function to Grp94Δ22-32 as shown in
The inventors next asked if mutation of each minor site alone could restore chaperone function in the context of an otherwise hyperglycosylated Grp94. Surprisingly, although blocking glycosylation at Asn107 restored the majority of the Grp94 chaperoning activity in the context of Grp94ΔPreN, MD-3xA, where the three MD minor sites were also blocked, the S109A mutation alone (Grp94ΔPreN,S109A) was completely nonfunctional. Of the other three minor sites, N445A was the only individual point mutant that was able to mature integrins, albeit at a lower level than WT, but none was capable of maturing TLR2 (
Although blockage of individual sites of glycosylation was largely unable to restore Grp94ΔPreN function, blockage of combinations of sites was able to restore activity. The double block mutant S109A/N445A restored a significant fraction of integrin expression, but only slightly restored TLR2 expression. When the inventors combined S109A and N445A with either N481A or N502A, the inventors found that the surface expression of both integrins and TLR2 is enhanced by N502A, but not N481A, suggesting that glycosylation at Asn481 may be functionally benign (
For the truncation constructs tested in this assay, the inventors found that the total levels of Grp94 were lower compared to WT when measured using a-Grp94 (9G10) intracellular staining on native protein by flow cytometry (
The Pre-N Domain is Highly Sequence Specific.
Removal of the first eleven residues of the Pre-N domain is sufficient to favor hyper-glycosylation and, as a result, impede chaperone function, indicating that the presence of this small region is critical.
In order to dissect the sequence specificity of the Pre-N Domain, the inventors engineered a set of alanine scan mutants of the approximately fifty (50) Pre-N domain residues. A schematic representation of alanine scan mutants of the Pre-N domain and Strand 1 (51) and into Helix 1 (H1) of the NTD tested by flow cytometry is shown in the left panel of
To confirm that these mutants abrogate Grp94 function by increasing deleterious N-linked glycosylation, the inventors mutated the minor N-linked sites and assayed this construct for chaperone function. Grp94 null pre-B cells were retrovirally transduced with Grp94 alanine scan mutants using the MigR1 vector, which co-expresses GFP. The expression of Grp94 clients (CD11a, CD49d, and TLR2) at the cell surface and the intracellular levels of Grp94 were analyzed by flow cytometry using protein-specific antibodies. Representative alanine scan constructs bearing mutations at N-linked glycosylation sequons were transduced and analyzed in the same manner. Representative histograms are displayed (
Hyper-Glycosylated Grp94 Forms Heterogeneous Oligomers that Retain ATPase Activity.
The preceding results show that hyper-glycosylation is deleterious to Grp94 chaperone function, but the mechanism by which the hyper-glycosylated chaperone loses function is unclear. To further investigate the potential causes of the observed loss of function, the inventors purified Grp94Δ22-32, which is fully glycosylated (
Surprisingly and unexpectedly, compared to WT unglycosylated Grp94, hgGrp94Δ22-32 does not migrate as a dimer but instead appears as a heterogeneous high molecular weight species with a significant fraction of the sample failing to enter the gel (
To further characterize the oligomeric hgGrp94 species, the inventors also analyzed its behavior using size-exclusion chromatography (SEC). As seen in
Heterogeneous populations of hgGrp94 are able to hydrolyze ATP. ATPase activity of hgGrp94 derived from mammalian cells or Grp94 derived from E. coli were measured using the fluorescence-based PiPer Phosphate assay (Thermo-Fisher). Control ATPase experiments indicate that S109A, N481A, and N502A mutations do not impact the hydrolysis activity of Grp94 (
N-glycan modeling analysis of Grp94 in the open state (PDB ID 201U) shows that Asn107, Asn481, and Asn502 are not found on loops, but, rather, at positions that are closely packed into the protein structure, which is uncommon for N-glycan sites. Glycosylation modeling was performed using the GlyProt server on PDB ID 201U. A monomer of the Grp94 dimer is shown for clarity (
Modeling of N-glycans in the closed dimer state of Grp94 (PDB ID 5ULS), on the other hand, reveals that only Asn217 and Asn481 can be modified (
Importantly, S109A, N481A, and N502A mutations had no impact on hydrolysis activity, confirming that these mutations did not lead to the observed ATPase activity for HEK293-derived proteins. Together, these data indicate that glycosylation at certain sites may alter the fold of Grp94 and lead to association between Grp94 molecules in an unknown fashion, but does not prevent ATP hydrolysis in the heterogeneous populations purified here.
This disclosure presents the results of an investigation of the role of the Pre-N domain in regulating total levels of Grp94 glycosylation and examined the effects of hyper-glycosylation on chaperone function and ATPase activity. Applicant's results indicate that when normal glycosylation is disrupted by high overexpression or mutations within the Pre-N domain, the chaperone becomes hyper-glycosylated with high glycosylation occupancy at all of the minor sites. Hyper-glycosylation renders the chaperone non-functional for client maturation, but function can be restored by mutating deleterious glycosylation sequons to prevent their modification. As the Pre-N domain is the N-terminal most portion of Grp94, the inventors speculate that this region may direct normal co-translational or post-translational folding and glycosylation during ER translocation. However, a molecular explanation behind how the intact Pre-N domain signals for normal glycosylation and therefore how the artificial lesions within this region tested here disrupt regulation remains unknown so far.
Two of the functionally deleterious sequons uncovered here, Asn107 and Asn445, are strictly conserved throughout hsp90 evolution due to their localization within ATP catalytic regions (Cherepanova et al., 2019). Asn107 is located on helix 2 of the NTD and directly interacts with bound nucleotides as described above, whereas Asn445 is part of the “catalytic loop” in the MD (Prodromou, 2016). Applicant's data show that these minor sites are heavily glycosylated when the Pre-N domain is truncated or absent. All other paralogs possess a shorter Pre-N extension, but retain the conserved sequons equivalent to Asn107 and Asn445 despite not being subject to N-linked glycosylation in their respective subcellular compartments. This observation suggests that one reason the other human paralogs may not functionally complement Grp94 is simply because the equivalent sequons are available for glycosylation. Analogous to Grp94 and the hsp90 family, BiP evolved as a result of gene duplication of an ancestral hsp70 gene. BiP, however, does not possess any N-linked glycosylation sequons nor does it contain a long extension from its N-terminus relative to cytosolic Hsp70. Thus there may exist an evolutionary correlation between the Pre-N domain and the need to self-regulate N-linked glycosylation.
Although Applicant's data indicate that small deletions of the Pre-N domain beyond the signal peptide are sufficient to hyper-glycosylate Grp94, the precise sequence downstream of this region is also highly important for maintaining chaperone activity. The observed specificity suggests that the nearly the entire length of the Pre-N domain, and not just residues 22-32, for example, may participate in specific interactions during co- or post-translational glycosylation of Grp94. As the signal peptide and the Pre-N domain are the first regions to be inserted into the ER, these regions may interact with components of the translocation or N-linked glycosylation machinery. As such, an unknown protein might recognize an extended surface or fold of the Pre-N domain early in translocation, leading to an interaction that ensures normal glycosylation is carried out. In support of this theory, the inventors found that wild type Grp94 is mono-glycosylated until an expression threshold has been reached. A hypothetical partner protein could become saturated at high Grp94 expression levels, which could then shunt a portion of newly synthesized Grp94 toward an alternate glycosylation pathway. Similar to the effect of saturating expression, truncation of the Pre-N domain led to a fundamental impairment of normal glycosylation, i.e. the critical interaction(s) cannot occur, and thus all newly synthesized mutant Grp94 becomes hyper-glycosylated. Alternatively, as pointed out by Qu et al., the usage of certain sites may be dictated by whether or not the specific site in question passes through the membrane co-translationally or post-translationally (Qu et al., 1994). In this way, the Pre-N domain and flanking regions may pause translation allowing the downstream amino acids to be translated and partially folded in the cytosol, which could lead the glycosylation machinery to skip the minor sites. A similar example is found in the N-terminal most portion of the simian rotavirus protein, VP7, but not the closely related bovine VP7, where the signal peptide and residues 64111 delay translocation into the ER (Stirzaker et al., 1990).
In multicellular organisms, OST is a membrane embedded complex consisting of at least eight subunits (Mohorko et al., 2011) (Braunger et al., 2018). Higher eukaryotes possess two paralogs of the catalytic subunit, STT3A and STT3B, with each associating with shared subunits as well as additional unique subunits. A recent study found that deletion of STT3A in cells led to hyper-glycosylation of all five of the minor glycosylation sites of Grp94 (Cherepanova et al., 2019), a phenomenon that appears to phenocopy the effect of mutating the Pre-N domain. Initially, it might seem surprising that genetic deletion a catalytic subunit could lead to an increase in glycosylation levels of Grp94. However, the authors speculate that there is a particular mechanism that normally restricts access of Grp94 to STT3A, which skips the minor sites and normally glycosylates Asn217. In this model, with STT3A deleted, STT3B must glycosylate Grp94 and it presumably does so without skipping any of the minor sites. Undoubtedly, determining if the Pre-N domain interacts specifically with STT3A or other proteins in the OST complex is an important area of future inquiry.
The question persists as to whether the expression of hgGrp94 is a programmed biological mechanism or if it forms simply as an unintended consequence of overexpression left only to be degraded as rapidly as possible. Arguing for functional value in humans is the fact that vertebrates retain six glycosylation sites, yet orthologs in invertebrates such as C. elegans and D. melanogaster have only two or three sites. It remains unclear if certain sites can be selectively modified or if hyper-glycosylation occurs stochastically to indiscriminately shut off activity. Does the biological fate of certain multi- or hyper-glycosylated species differ? Dersh et al. found that, in general, hgGrp94 is degraded at a faster rate than mgGrp94 by an OS-9 mediated, lysosomal-like mechanism (Dersh et al., 2014b). Yet, they found that only ˜50% of hgGrp94 is degraded over a period of 10-24 hours using pulse chase, indicating that this process is not rapid. Applicant's data supports the notion that hgGrp94 might be prone to degradation, as the inventors consistently observed lower intracellular levels of Pre-N truncated Grp94 compared to WT by flow cytometry and also observed that purified hgGrp94 has atypical behavior by Native-PAGE and SEC. Since OS-9 is involved in ERAD, it is unclear why these two proteins associate and get degraded, but yet they do so via an ERAD-independent mechanism.
In summary, the data presented here show that the Pre-N domain, a divergent structural feature within the hsp90 family, controls the total levels of N-linked glycosylation of Grp94. Further, the inventors revealed that this unique mechanism negatively regulates Grp94 activity under conditions of high Grp94 overexpression. Applicant's findings may provide context for cellular conditions in which Grp94 is highly up-regulated, including ER stress and multiple distinct cancers (Lee, 2014). Intriguingly, a recent study found that Grp94 is glycosylated at Asn107 in ovarian cancer cells (Sun and Zhang, 2015), suggesting that a significant portion of the total Grp94 population is non-functional for client maturation. It would be interesting to determine if the non-functional population of Grp94 is beneficial or pathological in the context of disease. In the former scenario, hgGrp94 could hypothetically help maintain ER proteostasis by binding misfolded proteins and facilitating their degradation alongside its own.
Further detail to enable a person of ordinary skill in the art to make and use the techniques described herein is provided in the following references, which are incorporated herein by reference and constitute part of the present disclosure: Huck, J. D., Que, N. L., Hong, F., Li, Z., and Gewirth, D. T. (2017a). Structural and Functional Analysis of Grp94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site. Cell Rep 20, 2800-2809; Huck, J. D., Que, N. L., Hong, F., Li, Z., and Gewirth, D. T. (2017b). Structural and Functional Analysis of Grp94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site. Cell reports 20, 2800-2809.
This application is a utility application filed under applicable portions of 35 U.S.C. § 111 and 37 CFR § 1.53, which claims priority to U.S. Provisional Application No. 62/984,545, filed on Mar. 3, 2020. The entire contents of the foregoing application is expressly incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62984545 | Mar 2020 | US |
