The Role of the C1 and C2 A-domains in Type VI Collagen Assembly*

Stephen G. Ball, Clair Baldock, Cay M. Kielty, and C. Adrian ShuttleworthDagger

From the University of Manchester, School of Biological Sciences, Wellcome Trust Centre for Cell/Matrix Research, 2.205 Stopford, Manchester M13 9PT, United Kingdom

Received for publication, April 4, 2000, and in revised form, September 25, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constructs of each of the three chains of type VI collagen were generated and examined in an in vitro transcription/translation assay supplemented with semipermeabilized cells. Each of the constructs when used in the in vitro system was shown to be glycosylated and to undergo intracellular assembly, the extent of which was determined by the nature of the C-terminal globular domains. All three chains containing the C1 domain formed monomers; however, the C2 domain was required for dimer and tetramer formation. In the case of the full-length alpha 2(VI) chain, monomers, dimers, and tetramers formed in a time-dependent manner. Although the splice variant alpha 2(VI)C2a could form monomers, it was unable to form dimers and tetramers. Similar results to the alpha 2(VI) chain were found for the full-length alpha 1(VI) chain, although assembly was at a slower rate. In the case of the alpha 3(VI) chain containing both C1 and C2 domains only monomers were observed. Addition of the C3, C4, and C5 did not change this pattern. Homology modeling suggested that a 10-amino acid insertion in the C2 domain of the alpha 3(VI) chain may interfere with dimer formation. A near full-length construct of the alpha 3(VI) chain only formed monomers but was shown to facilitate tetramer formation in cotranslation experiments.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Type VI collagen appears to have a ubiquitous distribution throughout connective tissues, and although it has been suggested that it links cells and other matrix components, its precise role in the matrix has still to be determined. Mutations in each of the three genes of type VI collagen have been linked to Bethlem myopathy, a mild form of muscle weakness and wasting (1). It is unclear why mutations produce a muscle disease rather than a more generalized phenotype, but perhaps type VI collagen has some tissue-specific functions, as well as interactions that occur in all tissues.

Three different chains have been described for type VI collagen, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) (2-5). Each chain contains a short triple-helical region and globular extensions at the N and C terminus. The majority of noncollagenous domains have homology to von Willebrand factor A-domains, and these are found at both the N and C termini of all three chains. In the case of the alpha 3(VI) chain, ten such domains (N1 to N10) may be found at the N terminus (2-5). In contrast to most other collagens, type VI collagen undergoes some polymerization prior to secretion. Intracellularly, triple-helical monomers align in an antiparallel manner with the C-terminal globular domains attached to the adjacent helix by disulfide bonds. These dimers then align with their ends in register to form tetramers, which are the secreted form of the molecule. End to end accretion of tetramers leads to microfibril formation in the extracellular matrix (6).

Tissue extraction and biosynthetic studies have demonstrated that alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains occur in stoichiometric proportions in a 1:1:1 ratio (6-8). These early observations have been confirmed by using cell lines deficient in one of the chains (9) and by examining secretion and assembly of recombinant type VI collagen (10). In the case of recombinant expression studies, it was shown that, when cell lines were stably transfected with either the alpha 1(VI) or alpha 2(VI) chain, only single polypeptides were secreted. Cells transfected with all three chains, however, produced a pepsin-resistant type VI collagen (10). The human osteosarcoma cell line (SaOS-2), under certain growth conditions, is deficient in alpha 3(VI), and although these cells have an abundance of alpha 1(VI) and alpha 2(VI) mRNAs, collagen VI was not detected in the medium by 4 weeks of culture (9). Stable transfection of these cells with the alpha 3(VI) cDNA enabled formation of type VI collagen dimers and tetramers, which were secreted and deposited into an extensive network (9). These results suggest that the alpha 3(VI) chain contains the sequences that enable chains to associate in the endoplasmic reticulum and form a triple helix.

Triple helix formation in collagen involves chain association, registration, nucleation, and propagation (11). Proper chain selection and registration is initiated by association of the C-propeptide. Sequences have been defined for the fibrillar collagens in the C-propeptide that are essential for chain selection (12), and the NC1 domains of both type VIII and X collagens have been shown to be critical in chain association (13, 14). By analogy, it is likely that the C-terminal domains in the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains of type VI collagen play essential roles in chain association and selection.

We have been interested in understanding what regulates and controls chain association in type VI collagen and have produced constructs of the human alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, which contain the N1 and C1 with and without the C2 domains, and a near full-length alpha 3(VI) chain and have used them in an in vitro transcription/translation system supplemented with semipermeabilized cells. This system has been used successfully to show correct folding of correctly aligned triple helices (15), to identify molecular recognition sequences that determine chain association (16), and to highlight the importance of C-terminal (NC1) sequences to short chain collagens (14). Our results show that the recombinant alpha 1(VI) and alpha 2(VI) chains can, indeed, form triple-helical monomers, and subsequently dimers and tetramers when the C2 domains were present, but not when only the C1 domain was present. Interestingly, the alpha 3(VI) chains could form monomers but were unable to form dimers and tetramers, although they were able to facilitate dimer and tetramer assembly in cotranslation experiments.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of alpha -Chain Constructs-- Nine alpha -chain cDNA constructs were generated by reverse transcription followed by overlap PCR1 (Fig. 1). Human placental total RNA (1 µg) was reverse transcribed with an oligo(dT) primer for 90 min using an Expand reverse transcription kit (Roche Diagnostics, Lewes, United Kingdom), according to the manufacturers instructions. Overlap extension PCR was performed using an Expand high fidelity PCR kit (Roche Diagnostics, Lewes, UK). Reaction mixtures (50 µl) consisted of ~10 ng of template cDNA, reaction buffer containing 1.5 mM MgCl2, 200 µM dNTPs, 50 pmol of forward and reverse primers, and 2.6 units of DNA polymerase mixture (thermostable Taq and Pwo). The reaction mixture was incubated for 3 min at 94 °C, immediately followed by 25 cycles of 1 min at 94 °C, 2 min at 60 °C, and 2 min at 72 °C and then incubated for 7 min at 72 °C. Each forward alpha -chain primer represented a chimera of the last 19 bases at the 3' end of the signal peptide sequence (CAGGCTCATTCAGGCTGGT), and the remainder were based on the start of the alpha -chain construct. In the case of the alpha 1(VI) and alpha 2(VI) chain sequences, the initial ATG start codon was omitted from each primer sequence. Each reverse alpha -chain primer contained either the endogenous or inserted stop codon (shown in below in bold face).



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Fig. 1.   Recombinant alpha -chain cDNAs used in this study. Nine type VI collagen alpha -chain constructs were generated, each containing the same signal peptide sequence, designated SP. N- and C-terminal domains are shown as boxes, whereas the helical region is represented by hatched lines. The estimated molecular size of each translation product is given, together with the number of base pairs (bp) of each construct, shown in brackets.

Generation of alpha 1(VI) Chain Constructs-- The alpha 1N1 forward primer (5'-CAGGCTCATTCAGGCTGGTAGGGCGGCCCGTGCTCTGC-3') (bases 52 to 70; see Ref. 4) and alpha 1C1 reverse primer (5'-GGGCAGGTGTACTATGGACACTTC-3') (bases 2487 to 2510; see Ref. 4) were used to amplify an expected fragment of 2458 base pairs (alpha 1C1; Fig. 1), whereas the alpha 1N1 forward primer and alpha 1C2 reverse primer (5'-GCAGGGTGGGCTAGCCCAGCGCC-3') (bases 3123 to 3145; see Ref. 4) generated an expected fragment of 3093 base pairs (alpha 1C2; Fig. 1).

Generation of alpha 2(VI) Chain Constructs-- The alpha 2N1 forward primer (5'-CAGGCTCATTCAGGCTGGTCTCCAGGGCACCTGCTCCG-3') (bases 25 to 43; see Ref. 4) and alpha 2C1 reverse primer (5'-CACGGACAGCTATGTTTGGCAGGG-3') (bases 2467 to 2490; see Ref. 4) were used to amplify an expected fragment of 2465 base pairs (alpha 2C1; Fig. 1), and the alpha 2N1 forward primer and alpha 2C2 reverse primer (5'-CGGCGGCGCTAGCAGATCCAGCGG-3') (bases 3063 to 3086; see Ref. 4) amplified a product of 3061 base pairs (alpha 2C2; Fig. 1), whereas the alpha 2N1 forward primer and alpha 2C2a reverse primer (5'-GCCCGGGCTTTAGCAGAGGGCCAGCGCGGTCTCGGGGT-3') (bases 3040 to 3077; see Ref. 21) generated an expected fragment of 2759 base pairs (alpha 2C2a; Fig. 1).

Generation of alpha 3(VI) Chain Constructs-- The alpha 3N1 forward primer (5'-CAGGCTCATTCAGGCTGGTGCTTGTAATCTGGATGTGATTCTGGGG-3') (bases 5131 to 5157; see Ref. 5) and alpha 3C1 reverse primer (5'-CCACAGGATGGCTAGATGTTGCAGATG-3') (bases 7398 to 7424; see Ref. 5) generated a fragment of 2293 base pairs (alpha 3C1; Fig. 1). The alpha 3N1 forward primer and alpha 3C2 reverse primer (5'-GGTTACAGGCTATGTTGTGGTGG-3') (bases 8282 to 8304; see Ref. 5) amplified a product of 3173 base pairs (alpha 3C2; Fig. 1), whereas the alpha 3N1 forward primer and alpha 3C5 reverse primer (5'-CGCTTAGGTTCCCATCACACTG-3') (bases 9144 to 9165; see Ref. 5) were used to generate an expected fragment of 4034 base pairs (alpha 3C5; Fig. 1). A alpha 3N8 forward primer (5'-CAGGCTCATTCAGGCTGGCTGGTTCATTGAAGTCAACAAGAGAG-3') (bases 944 to 964; see Ref. 5) and alpha 3C5 reverse primer were used to generate a fragment of 8221 base pairs (alpha 3N8; Fig. 1).

For each of the nine alpha -chain constructs, a signal peptide sequence of the alpha 1(VIII) collagen chain and ~200 base pairs of a 5'-untranslated sequence, known to promote efficient translation using semipermeabilized cells (14), was generated by overlap extension PCR from clone pBlu-Hal8. The signal peptide forward primer (5'-CGGGCACACTAAGAGTCAGC-3'), corresponding to the 5' end of the untranslated region, was used in each case, whereas the signal peptide reverse primer (5'-alpha -chain sequence-ACCAGCCTGAATGAGCCTG-3') represented a chimera consisting of between 9 and 34 bases of the 5' end of a specific alpha -chain construct and the last 19 bases at the 3' end of the signal peptide sequence. The signal peptide primers generated an expected fragment of ~300 base pairs for each alpha -chain. Purified PCR products from an alpha -chain construct reaction and a corresponding signal peptide reaction were combined in a second round overlap extension PCR. Reactions were performed in the absence of primers for six thermal cycles of 30 s at 94 °C, 1 min at 55 °C, and 30 s at 72 °C, to allow self-priming of the product overlap regions. Subsequent addition of the signal peptide forward primer and the appropriate alpha -chain reverse primer for 25 cycles of 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C allowed amplification of the alpha -chain construct. Each alpha -chain construct was purified and cloned into TA vector pGEM (Promega, Southampton, UK), except for the alpha 3N8 chain construct, which was cloned using an Expand cloning kit (Roche Diagnostics, Lewes, UK). The sequence identity and reading frame integrity were confirmed by dye-terminator automated sequencing.

Preparation of Semipermeabilized Cells-- HT-1080 human fibrosarcoma cells were maintained in Dulbecco's modified Eagle's medium containing sodium pyruvate (0.11 g/liter) and pyridoxine, supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin/penicillin, and 10% (v/v) fetal calf serum (Life Technologies, Inc., Paisley, UK). Confluent HT-1080 cells from a 75-cm2 flask were washed with phosphate-buffered saline, trypsinized using 2.5 mg/ml trypsin for 3 min and then resuspended in 8 ml of ice-cold KHM buffer (100 mM potassium acetate, 20 mM Hepes, pH 7.2, 2 mM magnesium acetate) containing 300 µg/ml soya bean trypsin inhibitor (Sigma). Cells were isolated by centrifugation at 280 × g for 3 min and then resuspended in 6 ml of KHM buffer containing 40 µg/ml digitonin (Calbiochem, Nottingham, UK) and incubated on ice for 5 min. Permeabilization was terminated by adding 8 ml of KHM buffer, isolating the cells by centrifugation, resuspending in 11 ml of 50 mM Hepes, pH 7.2, 90 mM potassium acetate, and incubating on ice for 10 min. Semipermeabilized cells were isolated by centrifugation and resuspended in 200 µl of KHM buffer, and endogenous mRNA was removed by adding CaCl2 to a concentration of 1 mM and monococcal nuclease (Sigma) to 10 µg/ml and incubating at room temperature for 12 min. After terminating the reaction by adding EGTA to a concentration of 2 mM, semipermeabilized cells were pelleted and resuspended in 100 µl of KHM buffer.

Transcription and in Vitro Translation-- Type VI collagen cloned cDNA was linearized downstream to the alpha -chain insert using SpeI (for alpha 1C1, alpha 2C1, alpha 2C2, alpha 3C1, alpha 3C2, alpha 3C5, and alpha 3N8) and PvuI (for alpha 1C2 and alpha 2C2a). Transcription was performed for 4 h at 37 °C in a reaction mixture containing 100 units of T7 RNA polymerase (for alpha 1C1, alpha 2C1, alpha 2C2, alpha 3C1, alpha 3C2, alpha 3C5, and alpha 3N8) or SP6 RNA polymerase (for alpha 1C2 and alpha 2C2a) (Promega, Southampton, UK), 1× transcription buffer, 10 mM dithiothreitol, 40 units of RNase inhibitor, and 3 mM NTPs (Roche Diagnostics, Lewes, UK). RNA was purified using spin columns (Qiagen, Crawley, UK) and eluted in 50 µl of RNase-free water containing 1 mM dithiothreitol and 20 units of RNase inhibitor.

Translations were carried out using a rabbit reticulocyte lysate system (Promega, Southampton, UK) for 15 min to 4 h at 30 °C. Each reaction contained 35 µl of lysate, 50 µg/ml sodium ascorbate, 50 mM KCl, 20 µM amino acids (minus methionine), 25 µCi of [35S]methionine, 40 units of RNase inhibitor, 2.5 µl of transcribed RNA (preheated to 60 °C for 10 min and then chilled on ice), and 4 × 105 semipermeabilized HT-1080 cells. Translation was terminated by addition of cycloheximide to a concentration of 1 mM, and reaction mixtures were immediately placed on ice. CaCl2 was added to a concentration of 10 mM, and reaction mixtures were digested with 250 µg/ml proteinase K (Sigma, Poole, UK) for 20 min. Digestion was stopped by adding 10 mM phenylmethylsulfonyl fluoride (Sigma, Poole, UK) and incubating on ice for 5 min and then N-ethylmaleimide (Sigma, Poole, UK) was added to a concentration of 25 mM and incubated on ice for 15 min. Cells were then washed by addition of KHM buffer and isolated by centrifugation three times at 14,000 × g for 4 min and was then resuspended in an appropriate buffer for subsequent digestion, immunoprecipitation, or direct SDS-PAGE analysis.

Pepsin Digestion-- Immunoprecipitates were resuspended in digestion buffer and incubated on ice for 10 min. The solution was then acidified with HCl (100 mM). Pepsin (300 µg/ml; Sigma, Poole, UK) was added, and samples were digested at 30 °C for 2 h. Digestion was stopped by neutralization with Tris base (100 mM) and addition of SDS-PAGE sample buffer.

Endoglycosidase H Digestion-- Isolated cells were resuspended in 100 mM Tris-HCl, pH 8.0, 1% SDS, 1% beta -mercaptoethanol and boiled for 5 min. Samples were centrifuged at 14,000 × g for 5 min to remove insoluble material and then an equal volume of 150 mM sodium citrate, pH 5.5, was added and incubated with 1 unit of endoglycosidase H (Roche Diagnostics, Lewes, UK) at 37 °C for 16 h. The reaction was terminated by adding SDS-PAGE sample buffer. Immunoprecipitates were treated in an identical manner except that no reducing agent was used.

Immunoprecipitation-- Isolated cells were resuspended in 1 ml of NET buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.25% (w/v) gelatin, 0.05% (v/v) Nonidet P-40, and 0.02% (w/v) sodium azide) and incubated on ice for 30 min. Samples were centrifuged at 14,000 × g for 10 min to remove insoluble material and then precleared at 4 °C for 1 h with 40 µl (10% (v/v)) of protein A-Sepharose. After centrifugation at 800 × g for 3 min, the supernatant was immunoprecipitated at 4 °C for 16 h with 20 µl of type VI collagen polyclonal antibody VIA or VIB (kindly donated by Dr. S. Ayad) (18). Samples were then incubated at 4 °C for 2 h with 80 µl (10% (v/v)) of protein A-Sepharose before the immunoprecipitates were isolated by centrifuging at 800 × g for 3 min. Immunoprecipitates were washed in 1 ml of NET buffer without gelatin or Nonidet P-40 and isolated three times and then resuspended in SDS-PAGE sample buffer.

SDS-PAGE Analysis-- Samples were resuspended in SDS-PAGE sample buffer in the presence or absence of 5% beta -mercaptoethanol and boiled for 5 min prior to analysis (17). Type VI collagen chains were analyzed using 8% (w/v) polyacrylamide gels, whereas monomers, dimers, and tetramers were analyzed using 3% (w/v) polyacrylamide 0.4% (w/v) agarose composite gels under nonreducing conditions. Gels were fixed, dried under vacuum, and imaged using a Fujix BAS 2000 phosphorimager.

Determining Chain Composition of Assemblies-- Directly after electrophoresis, individual assemblies were excised from a composite gel and incubated in 125 mM Tris-HCl, pH 6.8, 2% SDS, and 5% beta -mercaptoethanol at 4 °C for 16 h. Gel slices and the solution containing SDS-PAGE sample buffer were then loaded onto an 8% SDS-PAGE gel, and the protein components were electroeluted and resolved. The gel was fixed, dried under vacuum, and imaged using a Fujix BAS 2000 phosphorimager.

Homology Modeling-- The amino acid sequence of the C2 domain from the alpha 2 chain of human type VI collagen was used to probe the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) using the advanced BLAST 2.0 search (19). Sequences resulting from this search were the human von Willebrand factor A3 domain (vWFA3), the I domain from integrin CR3 (CD11a), and human von Willebrand factor A1 domain (vWFA1), which are all A-domains. The sequence with the highest homology was the vWFA3 domain with 22% identity to the C2 domain, and this structure was used as the starting point for homology modeling. Multalin (20) was initially used to align the sequences and then the secondary structural elements from the vWFA3 domain and the predicted secondary structure for the C2 domains (calculated using Predict Protein; see Ref. 21) were overlaid, and the sequence alignment was improved manually to produce a structure-based alignment. Most of the secondary structural elements would appear to be conserved based on the comparison of predicted and actual secondary structural elements.

All molecular modeling was performed on an R10,000 O2 Silicon graphics work station using QUANTA® and CHARMm 23.2® programs. The three-dimensional model of the C2 domain from the alpha 2 chain of type VI collagen was built based on the coordinates of the vWFA3 domain (PDB entry 1ATZ (22)). An homology model was built by copying the coordinates of the backbone of the vWFA3 domain and coordinates of completely conserved residues in the C2 domain. The remaining side chains were built in the Protein Design module using the Ponder and Richards' rotamer library. There were three insertions in the C2 domain sequence compared with the vWFA3 sequence; these were 5 residues in the loop between first alpha -helix and the second beta -strand, 1 residue in the loop following the third alpha -helix, and 1 residue in the loop between the fourth beta -strand and the fourth alpha -helix.

The C2 domain was energy minimized using steepest descents followed by the conjugate gradient algorithm to convergence removing bad steric and electrostatic contacts. The Protein Health module in QUANTA was used to check the integrity of the model using a Ramachandran plot and to identify buried hydrophilic or exposed hydrophobic residues and close contacts.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Translocation and Glycosylation of Products-- Short-length constructs consisting of the N1 domain, helical region, and C1 domain of each of the three alpha -chains of type VI collagen were translated in the presence of semipermeabilized cells and analyzed on SDS-PAGE gels. The molecular masses of the translation products of the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chain constructs were predicted to be 85, 86, and 80 kDa, respectively. Products of slightly higher molecular mass, relative to globular molecular mass markers, were observed (Fig. 2, lanes 1, 3, and 5). These bands were not susceptible to proteinase K treatment, confirming their translocation into the cellular rough endoplasmic reticulum (data not shown), and only translated products derived from proteinase K-treated cells were used in subsequent analysis.



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Fig. 2.   Glycosylation of translation products. Short-length constructs of each alpha -chain are shown. alpha 1C1, alpha 2C1, and alpha 3C1 were translated, and isolated cells were treated with proteinase K and analyzed directly (lanes 1, 3, and 5) or treated with endoglycosidase H (lanes 2, 4, and 6). Samples were analyzed by SDS-PAGE on an 8% gel under reducing conditions and visualized by phosphorimager analysis. Sizes of globular molecular mass markers are shown.

All three short-length alpha -chains were post-translationally glycosylated (Fig. 2, lanes 2, 4, and 6), as demonstrated by their faster migration after digestion with endoglycosidase H. The translation product of ~200 kDa (Fig. 2, lanes 1-6) was derived from the rabbit reticulocyte lysate, because it was translated in the absence of an RNA template or cells (data not shown).

Intracellular Assembly of Type VI Collagen-- Type VI collagen monomers undergo intracellular assembly into disulfide-linked dimers and tetramers prior to secretion. These assemblies are too large to monitor by SDS-PAGE gels, so low percentage agarose/acrylamide gels were used to examine formation of monomers, dimers, and tetramers. After transcription/translation of constructs coding for each alpha -chain for various lengths of time, products were immunoprecipitated using type VI collagen polyclonal antibody VIA or VIB and analyzed on nonreducing agarose/acrylamide composite gels. Size estimates of monomers, dimers, and tetramers formed following in vitro translation in the presence of semipermeabilized cells were obtained by comparison to monomers and dimers isolated from labeled immunoprecipitated cultured fibroblast medium (Fig. 3, lane 1).



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Fig. 3.   Agarose electrophoresis of translated products of alpha 1C1, alpha 2C1, and alpha 3C1. Short-length constructs of each alpha -chain, lacking the C2 domain (alpha 1C1 (lane 2), alpha 2C1 (lane 3), and alpha 3C1 (lane 4), were translated for 4 h. Translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from 35S-labeled cultured fibroblast medium, as shown in lane 1.

Comparison of the products from transcription/translation of constructs containing the N1 and C1 of each chain showed that material corresponding to monomers (molecular mass about 330,000) could be detected for each chain (Fig. 3, lanes 2-4). When constructs containing both the C1 and C2 domains were tested, additional bands were evident. In the case of the alpha 2(VI) chain, bands corresponding to both dimers and tetramers were seen after 4 h of translation (Fig. 4, lane 2). The C2 domain of this alpha -chain can undergo alternative splicing to produce alpha 2C2a; however, when this latter construct was translated for the same period and analyzed on agarose/acrylamide composite gels, no dimers were detected (Fig. 4, lane 3). Direct translation of the full-length alpha 2(VI) chain for 4 h in the absence of cells failed to reveal any dimers or tetramers (Fig. 4, lane 1), indicating that the formation of dimers and tetramers was cell-directed.



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Fig. 4.   Formation of alpha 2(VI) chain cell-free translation assemblies. Three different alpha 2(VI) chain constructs were translated for 4 h, alpha 2C1 lacking the C2 domain (lane 4), alpha 2C2a containing a variant C2 terminal domain (lane 3), and alpha 2C2, the full-length alpha 2(VI) chain containing the C2 domain (lane 2). The alpha 2C2 construct was also translated for 4 h in the absence of cells (lane 1). Translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium.

That these assemblies were glycosylated and formed from triple-helical monomers was shown by treatment with endoglycosidase H or pepsin, respectively (Fig. 5). Treatment of the immunoprecipitate from a full-length alpha 2(VI) chain translation with endoglycosidase H showed that the lower of the two monomer bands was unglycosylated (Fig. 5, lane 2). Pepsin digestion showed that triple-helical monomers were formed, and a protease-resistant fragment, susceptible to endoglycosidase H treatment, was apparent (Fig. 5, lanes 3 and 4, respectively).



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Fig. 5.   Deglycosylation and pepsin digest of alpha 2C2 translation product. The full-length alpha 2(VI) (alpha 2C2) chain construct was translated for 2 h and then terminated by the addition of 1 mM cycloheximide and 25 mM NEM. Isolated cells were treated with proteinase K and analyzed directly (lane 1), subjected to digestion with endoglycosidase H (lane 2), digested with pepsin (lane 3), or digested with pepsin followed by endoglycosidase H treatment (lane 4). Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium and a prestained SDS-PAGE 200-kDa myosin standard.

To look more closely at the rate of formation of intracellular assemblies, the full-length alpha 2(VI) chain construct was translated and analyzed on composite gels at various time points (Fig. 6B). Assemblies corresponding approximately to the molecular mass of alpha 2(VI) dimers (660 kDa) were observed after 2 h (Fig. 6B, lane 6) and to tetramers (1320 kDa) after 4 h (Fig. 6B, lane 8).



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Fig. 6.   Time course of formation of alpha 1(VI), alpha 2(VI), and alpha 3(VI) assemblies. A, the full-length alpha 1(VI) (alpha 1C2) was translated for between 15 and 240 min (lanes 1-8) before translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium. B, the full-length alpha 2(VI) (alpha 2C2) chain construct was translated for between 15 and 240 min (lanes 1-8) before translation was terminated by the addition of 1 mM cycloheximide and disulfide bond formation stopped by the addition of 25 mM NEM. After treating isolated cells with proteinase K, proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium. C, the alpha 3(VI) (alpha 3C2) chain construct containing the C2 domain was translated for between 15 and 240 min (lanes 1-8) before translation was terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium. D, equal aliquots (1 µl) of alpha 1C2, alpha 2C2, and alpha 3C2 RNA were cotranslated for between 15 and 240 min (lanes 1-8) before translations were terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIB, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Molecular mass sizes were estimated from monomers and dimers immunoprecipitated from cultured fibroblast medium.

Using a full-length alpha 1(VI) chain construct, dimers were observed after 90 min (Fig. 6A, lane 5), but tetramers were barely visible after 4 h (Fig. 6A, lane 8). Monomers only were observed using the alpha 1(VI) construct lacking the C2 domain (see above). In the case of the alpha 3(VI) construct containing C1 and C2, only monomers were observed by 4 h (Fig. 6C). The addition of the C3, C4, and C5 domains of the alpha 3(VI) chain to this construct made no difference in tetramer formation (data not shown).

Cotranslating equal amounts of RNA derived from the alpha 1(VI), alpha 2(VI), and alpha 3(VI) constructs containing both the C1 and C2 domains produced assemblies corresponding to monomers, dimers, and tetramers over the 4-h period (Fig. 6D). These assemblies were susceptible to reducing conditions indicating that they were stabilized by disulfide bonding (data not shown).

Rate of Formation of Dimers and Tetramers-- To follow the time course of formation of dimers and tetramers, agarose/acrylamide composite gels were scanned, and band intensity was determined by densitometry and then plotted against time (Fig. 7, A-D). These plots clearly showed that for the full-length alpha 1(VI) and alpha 2(VI) chains there is a linear increase in monomer with time and that, as this concentration increases, dimers and tetramers appear. In the case of the alpha 3(VI) construct containing C1 and C2 domains, although there is an increase in monomer with time, there is little evidence of dimer and tetramer formation during this time course.



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Fig. 7.   Rate of formation of monomers, dimers, and tetramers. The intensity of type VI collagen assemblies at various time periods (15 to 240 min) were evaluated by densitometry. The average densitometry values from three translations were calculated and then plotted against time. A shows alpha 1C2 translation assemblies, B shows alpha 2C2 translation assemblies, C shows alpha 3C2 translation assemblies, and D shows cotranslation (alpha 1C2/alpha 2C2/alpha 3C2) assemblies.

To examine how the alpha 3(VI) chain influenced type VI collagen assembly we cotranslated full-length alpha 1(VI) and alpha 2(VI) chains with a near full-length alpha 3(VI) chain (alpha 3N8; Fig. 1) lacking the N-terminal N10 and N9 domains and compared the rate of formation of monomers, dimers, and tetramers with those assembled, translating just full-length alpha 2(VI). These translations were performed using the same batch of semipermeabilized cells. Comparison of the alpha 2(VI) with cotranslation of alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains appeared to indicate more tetramer formation in cotranslations compared with single-chain translation (Fig. 8, compare lanes 8 and 4). That the tetramer in cotranslation experiments contained all three chains was shown after band isolation and SDS-PAGE in reduced conditions. Two bands, one corresponding to the alpha 1/alpha 2(VI) and the other to the alpha 3(VI) chain, were evident in the cotranslation, whereas only one corresponding to the alpha 2(VI) chain was found in the case of the alpha 2(VI) translation (Fig. 8, see insets). Translating the alpha 3N8 construct for 4 h produced only monomers (Fig. 8, lane 1). The influence of the alpha 3(VI) chain in cotranslation experiments was examined by quantification of the monomer, dimer, and tetramer bands by densitometry. This showed that dimers and tetramers were formed at a quicker rate when all three chains were present (Fig. 9). That the alpha 3(VI) chain was an important determinant of this increased formation was shown in cotranslation of both full-length alpha 1(VI) and alpha 2(VI) chains, where no difference in dimer or tetramer were evident compared with the individual chains (data not shown).



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Fig. 8.   Homo- and heteroassembly comparison. Using the same cell batch, translation was performed using the alpha 3N8 chain construct for 240 min (lane 1), the alpha 2C2 chain construct for various lengths of time (lanes 2-5), and a cotranslation using equal aliquots (1 µl) of alpha 1C2, alpha 2C2, and alpha 3N8 RNA for the same period (lanes 6-9). Translations were terminated by the addition of 1 mM cycloheximide and 25 mM NEM, and isolated cells were treated with proteinase K. Proteins were immunoprecipitated with type VI collagen polyclonal antibody VIA, analyzed on a nonreducing composite 0.4% agarose, 3% polyacrylamide gel, and visualized by phosphorimager analysis. Assemblies corresponding to tetramers, generated from an equivalent alpha 2C2 chain construct translation (as in lane 5) and a cotranslation (as in lane 9), were excised. Tetramers were reduced and then separated on an 8% SDS-PAGE gel, shown in boxes above the corresponding lane.



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Fig. 9.   Rate of homo- and heteroassemblies. The intensity of type VI collagen homoassemblies, shown in Fig. 8, lanes 2-5, and heteroassemblies, shown in Fig. 8, lanes 6-9, were evaluated by densitometry. Heteroassembly values were adjusted to the methionine content of three alpha 2C2 chain constructs and then the intensity values were plotted against time.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The semipermeabilized cells in conjunction with in vitro transcription/translation have been shown to recapitulate the early events of protein folding and assembly of multisubunit proteins destined for secretion (12, 14-16). The present results have shown for the first time that the alpha 1(VI) and alpha 2(VI) chains of type VI collagen can assemble intracellularly into triple-helical monomers and that dimers and tetramers subsequently form in a time-dependent manner. This is similar to the events that occur normally in the formation of type VI collagen, where tetramers are subsequently secreted from the cell and aggregate in the extracellular space end to end to form double-beaded microfibrils. The finding of defined molecular intermediates in the present study supports the contention that the semipermeabilized cells allow correct folding and chaperone association to occur and shows that dimer and tetramer formation occurs early in the secretory pathway of the cell. It is well known that chain selection and helix formation occur in the endoplasmic reticulum, but once sufficient monomers are present then dimer and tetramer formation follows.

A variety of studies have shown the importance of C-terminal interactions in chain selection and helix formation (11, 12, 14). In the case of type VI collagen, the implications from a number of studies were that alpha 3(VI) is an important determinant of chain association, although no information was available about the relative importance of the C1, C2, C3, C4, or C5 domains of this chain in that process. Interestingly, this study has shown that each of the three chains of type VI collagen can form monomers if the C1 domain is present, but the C2 domain appears to be essential to form higher molecular mass assemblies. The importance of the C2 domain to dimer and tetramer formation is shown dramatically for the alpha 2(VI) chain when the alternatively spliced C2 variant, C2a, was used. When the C2 was compared with the C2a variant it was clear that whereas constructs containing the full-length C2 could form dimers and tetramers, those containing the C2a variant could not. It has been suggested that production of alpha 2(VI) isoforms may be a mechanism for controlling the amount of mature type VI collagen (23). The present result would support this suggestion, as failure to produce dimers would result in recognition and removal by intracellular proteinases. Interestingly, the presence of the C2 domain in the alpha 3(VI) chain did not facilitate dimer and tetramer formation, which suggests that whatever sequence mediates antiparallel alignment in the C2 domains of the alpha 1(VI) and alpha 2(VI) chains is either not present or obscured in the alpha 3(VI) chain.

Structural studies have shown A-domains to be composed of a central beta -sheet flanked on each side by three alpha -helices, which is highly reminiscent of the Rossmann dinucleotide-binding fold (24, 25) The A-domains in type VI collagen have low (~15%) amino acid similarity to A-domains in other proteins, such as von Willebrand factor, complement, and integrin subunits. Identity between certain collagen VI A-domains is relatively high (47% for N7 and N9) but between others is much lower (18.5% for N1 and C1 of alpha 3(VI)).

Sequence studies have shown that the C1 domains of the alpha 1(VI) and alpha 2(VI) chains all have the residues in the metal ion-dependent adhesion site (MIDAS) sequence conserved, but this is not the case for C1 of the alpha 3(VI) chain. In the case of the C2 domain the alpha 1(VI) chain contains DXSXSTX, whereas the alpha 2(VI) chain contains DXSXXXD, although interestingly threonine is replaced by serine in this latter motif. No conservation of this sequence occurs in the alpha 3(VI) C2 domain. Metal ion coordination has been one means of generating protein-protein interactions, because it has been shown that the sixth coordination position of magnesium in the alpha -subunit of integrin CR3 is taken up by a carboxylate oxygen atom in another A-domain (24). However, the A3 domain of human von Willebrand factor binds collagen without metal binding (25) and highlights that there are other forms of A-domain interactions.

Of the A-domains for which there is high resolution structural data, the C2 domain of the alpha 1(VI) chain has most homology to the vWFA3 (Fig. 10). This domain also has a conserved MIDAS motif, but there is no evidence of metal ion binding, and this has been shown to bind to the collagen helix. It is interesting to speculate that binding occurs with the alpha 1(VI) C2 domain and the adjacent, antiparallel type VI triple-helical region when dimers form. Comparison of the models for the C2 domains of the alpha 1(VI) and alpha 2(VI) with that of the alpha 3(VI) suggest that there may be subtle changes in secondary folding that impact the ability of this domain to interact with collagen and hence form dimers (Fig. 11). In particular, there is a 10-amino acid insertion between the second and third beta -strand motifs in alpha 3C2 that may overlap and disrupt interactive residues at the MIDAS site (see Figs. 10 and 11).



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Fig. 10.   Sequence alignment of C2 domains of the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains compared with von Willebrand A3 domain. The secondary structure of von Willebrand A3 domain have been superimposed onto the sequence. alpha -Helices are colored yellow, and beta -strands are colored blue. For C2 domains the predicted secondary structure has also been superimposed. Residues involved in the MIDAS motif are shown in bold.



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Fig. 11.   A cartoon representation of the model of alpha 2C2. beta -Strands are shown as ribbons, and alpha -helices are shown as cylinders. 10-Amino acid insertion in the alpha 3C2 domain is sketched in blue.

The finding that alpha 1(VI) and alpha 2(VI) chains can form monomers, dimers, and tetramers suggests that one might expect to find these species secreted from cells and assembled into microfibrils. However, previous studies have suggested that although chains can be secreted, all three chains of type VI collagen are required for secretion and assembly of type VI collagen microfibrils (9, 10).

The literature is strewn with a veritable melange of reports on the fate of individual chains of type VI collagen. These range from stating that the alpha 1(VI) is secreted as a chain (26) to its being secreted as monomers, dimers, and/or tetramers (27). There are reports that the alpha 1(VI) can be found extracellularly in disulfide (28) or nondisulfide form (29). In SaOS-2 cells, which are deficient in alpha 3(VI) production, the majority, but not all, of the alpha 1/alpha 2(VI) is apparently retained in the cell, although chains did appear to get disulfide-bonded (9). In our hands immunoprecipitation of labeled medium from SaOS-2 cells showed a distinct monomer on agarose electrophoresis (data not shown). In contrast, following transfection of 293 cells with either alpha 1(VI) or alpha 2(VI) cDNAs, there was efficient secretion of polypeptide monomers and dimers (30).

Although transfection experiments may overload cellular function, it is surprising that there is so much variation and variability in the fate of single-chain transcripts or indeed why collagen appears to be able to avoid the cellular quality control mechanism (31). Early reports suggested that basal levels of collagen degradation were about 15% (32) and that as much as 30% of collagen was secreted from the cell in a nonhelical form (33). These results indicate that up to a third of unfolded collagen chains may not be removed intracellularly. Although these results were obtained looking at fibrillar collagens, data on type VI collagen (26-29) would suggest that a similar level of secretion occurs for this collagen. The present findings, showing that homotrimers can form into tetramers, suggests that these correctly folded assemblies escape quality control detection and are secreted. Their fate in the extracellular space is unknown, but findings with knockout mice would suggest that these assemblies are not as stable as heterotrimers, as no microfibrils are detected. Our cotransfection studies with all three chains of type VI collagen showed an increased rate of formation of dimers and tetramers and suggests, as previously reported, that the presence of alpha 3(VI) is an important determinant of type VI collagen formation.

The system used in the present study folds and assembles protein chains and shows clearly that, where alpha 1(VI) and/or alpha 2(VI) chains are present, the chains contain the information to allow formation of physiological monomers, dimers, and tetramers. We are currently investigating how these assemblies are handled in the cell and why homotrimer assemblies do not appear to form recognizable microfibrils.


    ACKNOWLEDGEMENTS

We are grateful to the Medical Research Council and Biotechnology and Biological Sciences Research Council for funding this work.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: University of Manchester, School of Biological Sciences, Wellcome Trust Centre for Cell/Matrix Research, 2.205 Stopford, Oxford Rd., Manchester M13 9PT, UK. Tel.: 0161-275-5079; Fax: 0161-275-5078; E-mail: ashuttle@fs1.scg.man.ac.uk.


    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MIDAS, metal ion-dependent adhesion site; NEM, N-ethylmaleimide.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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