Patent Application
20080166798
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
1. Field of the Invention
The invention relates to polypeptides capable of forming trimers. Methods of using such polypeptides are also disclosed.
2. Description of the Related Art
The type IIA amino (NH2)-propeptide is encoded by eight exons. The translated protein consists of a short globular domain, a 69 amino acid von Willebrand factor type C (VWfC) cysteine-rich domain, a minor collagen triple-helical domain containing 26 Gly-X-Y repeats and a short telopeptide domain which links the minor collagen domain to the major collagen triple-helix. Trimerization of most fibrillar collagens is dependent on the globular carboxy (COOH) propeptide for the recognition and association of the three polypeptide chains resulting in registered nucleation of triple-helix formation in a zipper-like fashion from the C- to N-terminus. Functions proposed for procollagen NH2-propeptides include the regulation of collagen fibrillogenesis and a feedback control of net collagen biosynthesis. It has also been proposed that the NH2-propeptide of type IIA procollagen regulates growth factor activity in the extracellular matrix.
Trimeric assembly of fibrillar NH2-propeptides affects protein valency and stability, which are important for function in vivo. This emphasizes the importance of a procollagen COOH-propeptide, or indeed other protein domains with similar function, to drive this trimerization process.
Pulmonary surfactant protein D (SP-D) is predominantly assembled as dodecamers, consisting of four trimeric subunits cross-linked by disulfide bonds. Each SP-D subunit contains an amino-terminal cross-linking domain, an uninterrupted triple-helical collagen domain consisting of 59 Gly-X-Y repeats, a trimeric coiled-coil neck domain and a C-type lectin carbohydrate recognition domain (CRD). Trimerization of SP-D subunits and subsequent oligomerization of these trimeric subunits to form higher order multimers, results in increased valency of the CRD, an essential pre-requisite for high affinity ligand binding. The neck domain of SP-D is the unit responsible for driving the trimerization of the three polypeptide chains of SP-D. It was demonstrated that a 35 amino acid sequence containing the human neck sequence was sufficient to form stable, non-covalent, trimeric complexes in vitro. The same sequence was found to be important for the association of the three CRDs of human SP-D; CRDs synthesized in prokaryotic cells without this neck domain were assembled as monomers.
The sequence of coiled-coil domains is characterized by a seven-residue repeat (commonly denoted (abcdefg)n) where positions a and d are primarily occupied by hydrophobic residues, positions e and g by charged residues, positions b, c and f by polar or charged residues, and n is an integer beginning with the numeral 1. The following Table 1 describes the hydrophobicity, polarity and charge of common amino acids:
The crystal structure of the neck and lectin domain of human SP-D has been solved and the coiled-coil region was visualized as a stretch of greater than 28 amino acids (Arg208-Pro235) consisting of approximately 8 helical turns.
Earlier work suggested that the presence of valine at the d positions favors the trimeric assembly of human SP-D. It was further suggested that the unusual fourth heptad, which contains Phe225 and Tyr228 in the a and d positions, respectively, might serve to initiate trimerization. However, no valine residues are found in the neck of rat SP-D. In addition, it was observed that deletion of the conserved fourth heptad repeat does not prevent trimerization of recombinant rat SP-D secreted by mammalian cells. On the other hand, internal deletions of residues 207-214 or 214-221 within the neck domain were found to block trimerization and indicated that sequences amino-terminal to Phe225 were required for trimerization.
The requirements for collagen trimerization and folding vary with the collagen type. Generally,.fibrillar collagens and type IV collagen require the presence of globular sequences C-terminal to the triple-helical domain to initiate chain registration. However, trimerization of type XII collagen is dependent on specific post-translational modifications of the collagen domain while chain association of the membrane-associated collagen, type XIII, occurs in the N-terminal region. Re-folding experiments on collagen type III indicated that inter-chain disulfide bridges at the C-terminus of the triple helix was sufficient to function as a nucleus for the re-folding of the triple helix. These findings suggest that the sequences requires for driving collagen trimerization can be manipulated as also exemplified by our ability to trimerize a procollagen amino propeptide using the a-helical coiled-coil domain of rat SP-D.
Two studies describe heterologous trimerization of collagen sequences to drive the trimerization of collagen sequences. Frank et al. (J. Mol. Biol. 308:1081-1089 (2001)) utilized the bacteriophage T4 fibritin foldon domain to synthesize a chimeric protein consisting of a synthetic collagen peptide (ProProGly)10 fused to the N-terminus of the foldon. The foldon domain, which consists of 27 amino acids and forms a β-propellar-like structure with a hydrophobic interior, was sufficient to drive the trimerization and correct folding of the synthetic collagen domain. Another study (Bulleid et al., EMBO J. 16:6694-6701 (1997)) showed that the COOH-propeptide of type III procollagen could be replaced with a transmembrane domain without affecting the folding of the collagen triple helix.
In addition, U.S. Pat. No. 6,190,886 to Hoppe et al. describes polypeptides comprising a collectin neck region, or variant or derivative thereof or amino acid sequence having the same or a similar amino acid pattern and/or hydrophobicity profile, are able to trimerize. Such polypeptides may comprise additional amino acids which may include heterologous amino acids, for example, forming a protein domain or derived from an immunoglobulin or comprising an amino acid which may be derivatized for attachment of a non-peptide moiety such as oligosaccharide, and may form homotrimers or heterotrimers. Heterotrimerization may be promoted by gentle heating, e.g. to about 50° C., then cooling to room temperature. One use for the polypeptides is in seeding collagen formation. Nucleic acid encoding the polypeptides and methods of their production are provided.
However, the trimerizing polypeptides described above are limited in their use because they are difficult to use to trimerize polypeptides with similar effect in vivo as well as in vitro. Because of this limitation, uses of the above trimerizing polypeptides in vitro do not accurately translate or cannot be used for therapeutic or other actions in vivo. In addition, the above described trimerizing polypeptides may not support normal folding of a procollagen propeptide domain, such a domain greatly enhancing the normal folding (folding found in vivo) of collagenous proteins both in vivo and in vitro. Additionally, many of the above described trimerizing polypeptides comprise a functional SP-D lectin domain which negatively affects the function of trimeric polypeptides in vivo.
Thus, what is needed is a minimum sequence of a trimerizing polypeptide capable of trimerizing procollagen propeptides to form collagenous molecules, and capable of trimerizing other oligomers, enabling use of such trimerizing polypeptides both in vitro and in vivo.
Accordingly, it is an object of the invention to overcome these and other problems associated with the related art. These and other objects, features and technical advantages are achieved by providing a minimum sequence for trimerizing procollagen propeptides and oligomers which take on the same comformation in vitro as in vivo.
This invention provides a method for trimerizing collagenous molecule monomers comprising the step of contacting a collagen domain and a non-collagenous trimerization domain. Preferably, the non-collagenous trimerization domain comprises a 14 amino acid sequence corresponding to the first two heptad repeats of the neck domain of mammalian pulmonary surfactant protein D. More preferably, the mammalian pulmonary surfactant protein D is rat pulmonary surfactant protein D. Altematively, the mammalian pulmonary surfactant protein D is human pulmonary surfactant protein D. Most preferably, the 14 amino add sequence is SEQ ID NO: 1.
In accordance with a further aspect of the invention, a method for trimerizing collagenous molecule monomers without a dimeric intermediate is provided comprising the step of contacting a collagen domain and a non-collagenous trimerization domain. Also provided is a method for producing a native conformation of NH2-propeptide of type IIA procollagen in vitro comprising the step of contacting a collagen domain and a non-collagenous trimerization domain.
In accordance with yet another aspect of the invention, a trimerized collagenous molecule monomers produced by contacting a collagen domain and a non-collagenous trimerization domain is provided. Additionally, a NH2-propeptide of type IIA procollagen produced by contacting a collagen domain and a non-collagenous trimerization domain is provided.
In accordance with yet another aspect of the invention, a polypeptide having the sequence of SEQ ID NO: 1 is provided. Further, a trimer comprising three collagenous molecule monomers is provided, said monomers consisting of a truncated SP-D domain of SEQ ID NO: 1. In one embodiment, a collagenous molecule monomer consisting of two heptad repeats of SP-D is provided, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 2. In another embodiment, a collagenous molecule monomer comprising two contiguous sites for BS3 cross-linking within the fourth heptad repeat of SP-D is provided, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 4. In yet another embodiment, a truncated fusion protein consisting of two heptad repeats of SP-D is provided, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 2. In yet another embodiment, a truncated fusion protein consisting of three heptad repeats of SP-D is provided, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 3.
A further aspect of the invention provides a chimeric gene construct comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding the neck domain and lectin domain of SP-D. Additionally, a chimeric gene construct comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding the neck domain of SP-D. Still further, a fusion protein comprising a IIA NH2-propeptide collagen domain and a 14 amino acid sequence of the SP-D coiled-coil neck domain of SEQ ID NO: 1.
In another aspect of the invention, a cell transfected with a chimeric gene construct is provided comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding the neck domain and lectin domain of SP-D. In another embodiment, a cell transfected with a chimeric gene construct is provided comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding the neck domain of SP-D. In addition, a stably transfected cell line is provided comprising a chimeric gene construct comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding the neck domain and lectin domain of SP-D. In yet another embodiment, a stably transfected cell line is provided comprising a chimeric gene construct comprising a cDNA encoding exons 1 through 8 of type IIA NH2-propeptide operably linked to a cDNA encoding SEQ ID NO: 1.
In another aspect of the invention, a polypeptide is provided wherein the first amino acid sequence is SEQ ID NO: 1. Further, a nucleic acid comprising a sequence of nucleotides encoding a polypeptide according to the above. Still further, a nucleic acid is provided wherein said nucleic acid further comprises a vector. In another aspect, a host cell containing a nucleic acid encoding a polypeptide having SEQ ID NO: 1 is provided. Preferably, a nucleic acid is provided, wherein the encoding sequence is operably linked to a regulatory sequence for expression of the polypeptide.
In yet another aspect of the invention, a host cell is provided containing the nucleic acid encoding a polypeptide having SEQ ID NO: 1. In a further aspect, a trimer is provided comprising the polypeptide having SEQ ID NO: 1. In one alternative, the trimer is a homotrimer. In another alternative, the trimer is a heterotrimer.
In another aspect of the invention, a protein expression method is provided comprising expressing a polypeptide having SEQ ID NO: 1 from a nucleic acid encoding the polypeptide. In yet another aspect of the invention, a polypeptide trimerizing method is provided comprising forming a trimer comprising a polypeptide having SEQ ID NO: 1 following its expression. In one altemative, the trimer is a homotrimer. In another altemative, the trimer is a heterotrimer.
These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, examples and appended claims.
The present invention is a short, amphipathic helical trimerizing polypeptide VASLRQQVEALQGQ (SEQ ID NO: 1) derived from the rat SP-D neck domain which can drive the trimerization of a fibrillar collagen NH2-propeptide as well as other propeptides and oligomers.
The present invention describes an efficient system for producing high levels of a correctly-folded NH2-propeptide of type IIA procollagen. This approach could likely be applied to the synthesis other procollagen NH2-propeptides, and other oligopeptides, which are difficult to isolate from tissues. Given that the propeptide is trimeric and correctly-folded, it will be possible to examine the contributions of valency to the biological function of this peptide. The ability to express a secreted trimeric propeptide without inclusion of the functional lectin domain of SP-D will also enable us to investigate the effects of the propeptide in in vivo models of tissue development and repair.
Such a trimerizing polypeptide results in a IIA NH2-propeptide which is folded in vitro the same as it is in viyo. The amino acid sequence consists of the first two heptad repeats of the neck domain, which is in agreement with our previous deletional mutagenesis studies showing that amino-terminal regions of the neck domain are important for initiating trimerization (Zhang et al., J. Biol. Chem. 276:19862-19870 (2001)). This is by far the shortest sequence found to permit trimerization of a collagenous molecule, and the first to demonstrate the use of a heterologous trimerization cassette to support the normal folding of a procollagen propeptide domain.
High levels of a correctly-folded IIA NH2-propeptide were produced using this system, which will enable the study its biological function in vitro. Establishing a minimum sequence of the SP-D neck domain that can drive tnimerization without inclusion of the functional SP-D lectin domain allows the study the function of the trimeric IIA propeptide in vivo. Knowledge gained from these findings may be applied to produce other procollagen propeptides or indeed other collagenous proteins for functional studies.
The polypeptide of the present invention is a 14 amino acid sequence derived from the first two heptad repeats of the α-helical coiled-coil domain of rat SP-D (SEQ ID NO:1). This polypeptide can drive the trimerization of a heterologous procollagen NH2-propeptide sequence. Although IIA propeptides alone are secreted as monomers, a IIA/SP-D chimera with a truncated SP-D neck domain terminating at residue 218 was sufficient to drive trimerization. Truncations at residue 211 or 203, containing one or no heptad repeats, respectively, were secreted as monomers. This is the shortest sequence ever described to support the trimerization of a collagen sequence.
In addition, trimerization is accompanied by folding of the collagen triple helical domain and that, following cleavage from the SP-D sequence, the IIA NH2-propeptide retains its trimeric conformation. Amino acid analysis revealed that approximately 80% of the potential proline residues in the Y position of the collagen sequence are hydroxylated, consistent with the formation of a stable triple helix. These levels of hydroxylation are comparable to that reported for the al chain of the NH2-propeptide of type I procollagen extracted from developing bone (Fisher et al., J. Biol. Chem. 262:13457-13463 (1987)). In addition, the melting temperature of the collagen helix within the recombinant propeptide was similar to other comparably hydroxylated collagens, approximately 42° C. It has been suggested that a subpopulation of IIA NH2-propeptide trimers that migrated as trimers on SDS-PAGE. In this regard, Fisher et al. reported that the natural type I NH2-propeptide is not efficiently denatured by SDS treatment prior to electrophoresis. Together, these findings indicate the synthesis of a stable, trimeric IIA NH2-propeptide nearly identical to that found in vivo.
The ability of a 14 amino acid sequence to direct trimerization is surprising. Previous studies have shown that a classical two heptad repeat coiled-coil sequence is unable to form an autonomous folding unit (Su et al., Biochemistry 33:15501-15510 (1994)). Even the complete neck domain of SP-D is short compared to many coiled-coil domains, which average 7 repeats or 14 helical turns for three-stranded coiled-coils. The potential importance of β-branched side-chains for determining the assembly of coiled-coils was emphasized by Harbury et al. (Science 262:1401-1407 (1993)). In that study the occurrence of β-branched residues at the “d” position disfavored dimers, while these residues at the “a” position disfavored tetramers, and the presence of branched residues at both positions favored trimers. Given the occurrence of valine residues in the first three “a” positions of the human SP-D neck sequence (
However, no β-branched amino acids occur in these positions in the rat sequence, SMLRQQMEALNGK (SEQ ID NO:2), and none of the other known SP-Ds or related collectins show a similar conservation of P-branched residues in this position (e.g., bovine SP-D, VNALRQRVGILEGQ, SEQ ID NO:3). Studies using model peptides and surveys of known coiled-coils have identified residues that favor various oligomeric states. Residues found in the “a” and “d” positions of SP-D are usually non-discriminatory with respect to oligomerization or favor dimer formation. For example, leucine, which is present in the “d” position of the first three heptad repeats of SP-D, marginally favors dimers over trimers. Consistent with these observations, analysis of both human and rat (-helical coiled-coil sequences using MultiCoil predicted a dimeric association. For example, dimer formation probability for the human SP-D coiled-coil sequence was approximately 90%, or 70% for the rat sequence, using the available windows of 21 residues.
Thus, it seems likely that other interactions contribute to the stability or oligomerization of the 14 amino acid sequence. In this regard, g-e′ ionic interactions can contribute to the stability and oligomerization of some α-helical coiled-coils. Although most discussions emphasize the effects of electrostatic interactions on stability, Beck et al. recently showed that specific electrostatic interactions were required for trimerization of the considerably longer coiled-coil domain of cartilage matrix protein. Inspection of the neck sequence of rat SP-D suggests the possible occurrence of an intra-helical ionic interaction (i to i+4 spacing between Arg208 and Glu212) and/or an inter-chain ionic interaction (i to i+5 spacing between Asp203 and Arg208; g-e′) (
In any case, the finding that mIIA-218 is secreted as monomers, while IIA-218 is secreted as trimers, shows that the collagen domain contributes to trimer stability. Thus, both the amino-terminal heptad repeats of the neck of SPD and the IIA collagen sequence are required to form stable chimeric trimers. This represents the direct demonstration of a cooperative and mutually-stabilizing interaction between a collagen domain and its non-collagenous trimerization domain.
The mIIA-237 fusion protein reproducibly trimerizes, but without a detectable dimeric intermediate. Trimerization was also more efflcient, requiring less cross-linker than for the other truncation mutants. We speculate that this “all-or-none” cross-linking of mIIA-237 results from the presence of two contiguous sites for BS3 cross-linking at Lys229-Lys230 within the fourth heptad repeat. Although this seems at odds with the observation that cross-linking of IIA/SP-ID also proceeds through a dimeric intermediate, the three chains may not be within an equivalent environment compared to the context of the intact neck+CRD domain.
The crystal structure of the human SP-D neck+CRD shows a striking deviation from 3-fold symmetry involving the fourth heptad repeat, with one of the three tyrosines at position 228 bured, and the other two partially exposed (Hakansson et al., Structure 7:255-264 (1999)). Thus, our findings are consistent with the possibility that asymmetry is imposed on the neck by the presence of the CRD domain. Another potential implication is that the observed asymmetry exists in solution, and is not simply an artifact of crystallization.
Any three identical or different polypeptides containing the neck-region may form homotrimers or heterotrimers under appropriate conditions. A homotrimer consists of three polypeptides which are the same. A heterotrimer consists of three polypeptides, at least two of which are different. All three polypeptides may be different. One, two or all three polypeptides in a heterotrimer may be a polypeptide according to the invention, provided each polypeptide has a region able to trimerize.
The present invention further provides nucleic acid comprising a sequence of nucleotides encoding a polypeptide able to form a trimer and comprising SEQ ID NO:1, an amino acid sequence variant thereof or derivative thereof, or a sequence of amino acids having an amino acid pattern and/or hydrophobicity profile the same as or similar to SEQ ID NO:1, fused to a heterologous sequence of amino acids, as disclosed herein. The nucleic acid may comprise an appropriate regulatory sequence operably linked to the encoding sequence for expression of the polypeptide. Expression from the encoding sequence may be said to be under the control of the regulatory sequence. Preferably, a variant, derivative or sequence having an amino acid pattern and/or hydrophobicity profile will follow the following formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 2. Although less preferable, a collagenous molecule monomer may comprise two contiguous sites for BS3 cross-linking within the fourth heptad repeat of SP-D, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 4. In addition, a truncated fusion protein may consist of two heptad repeats of SP-D, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 2. Finally, a truncated fusion protein consisting of three heptad repeats of SP-D may be provided, the heptad repeat having the formula:
(abcdefg)n
wherein positions a and d are occupied by hydrophobic residues; positions e and g by charged residues; positions b, c and f by polar or charged residues; and n is 3.
Also provided by the present invention are a vector comprising nucleic acid as set out above, particularly any expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell containing any such vector or nucleic acid.
A convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it. Accordingly, the present invention also encompasses a method of making a polypeptide according to the present invention, the method comprising expression from nucleic acid encoding the polypeptide, either in vitro or in vivo. The nucleic acid may be part of an expression vector. Expression may conveniently be achieved by growing a host cell, containing appropriate nucleic acid, under conditions which cause or allow expression of the polypeptide.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include HeLa cells, baby hamster kidney cells and many others. A common, preferred bacterial host is E. coli.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including. promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The relevant disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.
Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.
In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of a host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. Following expression, polypeptides may be caused or allowed to trimerize. This may be prior to or following isolation.
A method of seeding a collagenous triple-helix involves causing or allowing trimerization of such a polypeptide. It may involve first the production of the polypeptide by expression from encoding nucleic acid therefore. The present invention provides such nucleic acid, a vector comprising such nucleic acid, including an expression vector from which the polypeptide may be expressed, and a host cell transfected with such a vector or nucleic acid. The production of the polypeptide may involve growing a host cell containing nucleic acid encoding the polypeptide under conditions in which the polypeptide is expressed. Systems for cloning and expression, etc. are discussed supra, and are well known in the art.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following specific examples are offered by way of illustration and not by way of limiting the remaining disclosure.
In order to study the polypeptides and trimerization methods of the present invention, a chimeric gene construct was synthesized consisting of cDNA encoding full-length type IIA NH2-propeptide (exons 1-8;
Immunoblotting of the EDTA-eluted protein with anti-IIA, anti-IIE 3-8 or anti-SPD polyclonal antisera confirmed identification of IIA/SP-D. Results were identical with all three antibodies and
To investigate the structure of the recombinant IIA NH2-propeptide, IIA/SP-D fusion protein was digested with purified bacterial collagenase, and the major collagenase-resistant bands were characterized by N-terminal sequencing. SP-D, which contains its own collagen domain, was included as a control. As shown in
To confirm that the IIA NH2-propeptide contained a correctly-folded collagen triple helix, the propeptide purified by enterokinase cleavage of IIA/EK/SP-D was analyzed by circular dichroism (CD) spectroscopy. The CD spectrum of a collagen triple helix is characterized by a small positive peak at 220-225 nm, a crossover at 213 nm and a trough at approximately 197 nm (Goodman et al., Biopolymers 47:127-142 (1998)).
Chemical crosslinking was used to examine the state of oligomerization of the IIA collagen domain. In particular, crosslinking profiles were compared for: 1) the wild-type IIA/SP-D fusion protein, 2) the IIA NH2-propeptide purified after MMP-9 cleavage of the fusion protein, and 3) the IIA NH2-propeptide synthesized without fusion to the neck/CRD domains of SP-D. As shown in
The trimerization domain of rat SP-D is a coiled-coil structure that consists of four heptad repeats as depicted in
Based on published literature, it was shown that a two heptad repeat coiled-coil sequence cannot form an autonomous folding unit (Su et al., Biochemistry 33:15501-15510 (1994)). Thus, it is highly likely that co-operative interactions exists between the collagen domain and the short, 14 amino acid sequence of the SP-D trimerization domain to stabilize the truncated fusion protein (mIIA-218,
A chimeric construct was synthesized by linking the cDNA encoding the NHz propeptide of type IIA procollagen (
IIA/SP-D cDNA was excised from pGEM-3Z by EcoRI digestion and ligated into the multiple cloning site of a vector suitable for expression of the polypeptide in Chinese Hamster Ovary (CHO) cells (Ausubel et al., Current Protocols in Molecular Biology (Ausubel, R. M., Brent, R., Kingston, R. E., Moore, S. S., Seidman, J. G., Smith, J. A., and Struhl, K., Eds.), John Wiley & Sons, New York (2000)) distal to a cytomegalovirus promoter/enhancer and proximal to a glutamine synthetase gene. CHO cells (CHO-K1; ATCC CCL-61) were transfected with the ligated vector-IIA/SP-D using Lipofectamine (Invitrogen, Carlsbad, Calif.) and grown in selection Glasgow's minimum essential medium (GMEM; Invitrogen, Carlsbad, Calif.) containing 10% dialyzed FBS and the glutamine synthetase inhibitor, methionine sulfoxamine (MSX; 25-50 μM) for 2-3 weeks. Stable clones were obtained as described by Crouch and colleagues for the expression of recombinant rat-SPD (Crouch et al., J. Biol. Chem. 269:15808-15813 (1994)). To assess the importance of the trimerizing neck domain, a control vector construct was constructed consisting only of cDNA encoding full-length IIA NH2-propeptide, devoid of cDNA encoding the neck and lectin domains. This construct was used in transient transfections of CHO cells using Lipofectamine reagent.
Media from transiently transfected CHO cells were screened for the presence of the fusion protein by an enzyme linked immunoassay using rabbit anti-human exon 2 (IIA) antibody (Oganesian et al., J. Histo. Cytochem 45:1469-1480 (1997)), chicken IgY anti-human Exon 3-8 antibody or rabbit anti-rat SP-D antibody (Persson et al., J. Biol. Chem. 265:5755-5760 (1990)). Immuno-positive proteins labeled with rabbit-HRP secondary antibodies were detected by enhanced chemiluminescence using SuperSignal® chemiluminescent substrate (Pierce Chemical Co., Rockford, Ill.). Clones expressing the IIA/SP-D fusion protein were selected and cultured further by exposure to 50-100 μM MSX and resulting conditioned media was dialyzed against TBS, pH 7.5, containing 10 mM EDTA. CaCl2 (20 mM) was added to the dialyzed media and IIA/SP-D was subsequently purified by maltosyl-agarose chromatography (Church et al., supra). Because the interaction of the CRD with maltose is calcium-dependent (Persson et al., supra), IIA/SP-D was eluted from the column with TBS/10 mM EDTA, pH 7.5. Eluted fractions were analyzed by SDS-polyacrylamide gel electrophoresis, silver staining and Western blotting.
Bacterial collagenase was purified by gel filtration chromatography using crude collagenase as the starting material (Worthington Biochemical Corp., Lakewood, N.J.) (Peterkofsky et al., Biochemistry 10:988-994 (1971)). IIA/SP-D or rat SP-D (30 pig) in TBS/10 mM EDTA, pH 7.5, was digested with purified bacterial collagenase (1 μg) containing CaCl2 (20 mM) and N-ethylmaleimide (5 mM), overnight at 37° C. Fresh collagenase (1 μg) was added for a further 3 hours at 37° C. followed by EDTA (4 mM) to stop the reaction. An aliquot (5 μg) of digested and undigested IIA/SP-D or rat SP-D was electrophoresed through a 4-20% SDS-polyacrylamide gel to confirm collagenase digestion. The major collagenase-resistant products were detected by Coomassie blue staining and subjected to N-terminal amino acid sequencing. Collagenase-digested IIA/SP-D or SP-D was transferred to Sequi-Blot PVDF membrane (Bio-Rad, Hercules, Calif.), stained with Coomassie blue, excised and sequenced on an ABI 473A protein sequencer equipped with model 610A data analysis software.
Approximately 100 μg of wild-type IIA/SP-D fusion protein was digested overnight at 37° C. with human recombinant MMP-9 at an enzyme:substrate ratio of 1:100. MMP-9 cleaves within the telopeptide domain of the IIA propeptide on either side of Q157 and M174 (Persson et al., supra). Since MMP-9 has two cleavage sites within the telopeptide and cleavage is not always 100% efficient, we proceeded to synthesize a mutant IIA/SP-D chimeric construct containing an enterokinase cleavage site in the exon 8-encoded telopeptide. Using the QuikChangerm Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), oligonucleotide primers were designed to change the DNA sequence encoding amino acids 161-165 in exon 8 of the wild-type IIA NH2-propeptide (151GFDEK185) to one which encodes the EK cleavage site (116DDDDK165). Stable CHO cell lines producing this mutant fusion protein (IIA/EK/SP-D) were produced as described above. Approximately 0.001% w/w of enterokinase (New England Biolabs, Beverly, Mass.) was added to purified IIA/EK/SP-D protein overnight at room temperature.
Cleavage by MMP-9 or enterokinase was confirmed by gel electrophoresis, silver staining and immunoblotting using antibodies specific for the IIA (exon 2) domain or the CRD of SP-D. Cleaved products were calcified and applied to a maltosyl-agarose column to separate the IIA NH2-propeptide (present in the flow-through) from the neck/CRD of SP-D (present in the EDTA eluate).
Covalent cross-linking was performed using bis-(sulfosuccinimidyl)suberate (BS3; Pierce Chemical Co., Rockford, Ill.). Increasing amounts of BS3(0, 0.1, 0.5, 1 and 2 mM final concentration) prepared in 5 mM sodium citrate, pH 5, was added to each recombinant proteins for 1 hour at room temperature. Addition of SDS-PAGE loading buffer containing Tris-HCl (0.5 M) inhibited the reaction. Samples were boiled for 5 minutes prior to SDS-PAGE, which was carried out in the absence of sulfhydryl reduction. Cross-linked proteins were identified by silver staining or immunolocalization using anti-IIA (exon 2) polyclonal antisera.
Approximately 50 μg of IIA NH2-propeptide (0.2 mg/ml in PBS, pH 7.5), purified by cleavage of the mutant IIA/EK/SP-D fusion protein containing the enterokinase cleavage site, was analyzed by circular dichroism (CD) spectroscopy. A Jasco (Easton, Md.) J715 spectropolarimeter with a thermostated quartz cell, path length of 0.1 cm, was used and the spectrum was recorded at 5° C. between 180-260 nm. To determine the melting temperature of the IIA NH2-propeptide, the spectrum was monitored at 225 nm from 5° C. to 70° C.
Equilibrium sedimentation experiments were performed using a Beckman (Fullerton, Calif.) Optima XL-A analytical ultracentrifuge using a six-channel centerpiece in an AN-60 Ti rotor. IIA NH2-propeptide, purified from enterokinase cleavage of the IIA/EK/SP-D mutant protein, in PBS (pH 7.5) was analyzed at three concentrations: 0.2, 0.4 and 0.8 mg/ml. Experiments were performed at two speeds (20,000 and 28,000 rpm) at a temperature of 20° C. and wavelength of 280 nm. Data were fitted using WinNonlin® (Pharsight, Mountain View, Calif.) V1.035
(http://www.ucc.uconn.edu/˜wwwbiotc/UAF.html) and a partial specific volume of 0.73 cm3/g was used for determining the molecular weight.
To determine the minimum sequence that can function as a trimerizing unit, mutant constructs were designed containing termination codons at specific locations within the heptad repeats of the coiled-coil. Using IIA/SP-D cDNA in pGEM-3Z as a substrate, four mutant constructs were synthesized using the QuikChangem Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). The sequence of the mutant was confirmed by DNA sequencing. Mutant IIA/SP-D cDNA constructs were excised from pGEM-3Z by EcoRI digestion and sub-cloned into a vector suitable for expression of the polypeptide in CHO cells. Correct orientation of the mutant cDNA insert in the vector was confirmed by restriction enzyme digestion (Hindlil and Bglll, Promega, Madison, Wis.) and agarose gel electrophoresis. CHO cells were transiently-transfected with each mutant construct using FuGENE 6 reagent (Roche, Switzerland) according to the manufacturer's instructions. Proteins were precipitated from the conditioned medium overnight at 4° C. with 33% ammonium sulfate. Precipitated proteins were washed three times in saturated ammonium sulfate, resuspended in PBS and dialyzed overnight in cold PBS. Chemical cross-linking of each mutant protein was carried out as described above. Proteins were detected by SDS-PAGE and immunolocalization of Western blots using the anti-IIA polyclonal antibody.
To determine if the minor collagen domain of the IIA NH2-propeptide contributes to the stability of the truncated fusion protein, we generated a related truncation mutant with an associated deletion of the collagen sequence (mIIA-218,
The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.
All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention. Specifically referred to and included herein in its entirety is a publication by K McAlinden, et a/., entitled: Trimerizatfon of the amino propeptfde of type IIA procollagen using a 14-amino acid sequence derived from the coiled-coil neck domain of surfactant protein D. J Biol Chem. 277(43):41274-81 (2002).
This application claims priority from Provisional Application Ser. No. 60/497,054 filed on Aug. 22, 2003, which is incorporated herein by reference in its entirety.
This invention was made in part with Government support under NIH Grants AR-36994, HL-29594, and HL-44015. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2004/027381 | 8/21/2004 | WO | 00 | 3/18/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60497054 | Aug 2003 | US |
