The Role of the alpha 3(VI) Chain in Collagen VI Assembly
EXPRESSION OF AN alpha 3(VI) CHAIN LACKING N-TERMINAL MODULES N10-N7 RESTORES COLLAGEN VI ASSEMBLY, SECRETION, AND MATRIX DEPOSITION IN AN alpha 3(VI)-DEFICIENT CELL LINE*

Shireen R. LamandéDagger , Emanouil SigalasDagger , Te-Cheng Pan§, Mon-Li Chu§, Marie Dziadek, Rupert Timplpar , and John F. BatemanDagger **

From the Dagger  Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia, § Department of Dermatology and Cutaneous Biology and Department of Biochemistry and Molecular Pharmocology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541,  Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052, Australia, and par  Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha 1(VI) and alpha 2(VI) chains have also been proposed. To address this question directly and study the role of the alpha 3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha 3(VI) production. Northern analysis showed an abundance of alpha 1(VI) and alpha 2(VI) mRNAs, but no detectable alpha 3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha 3(VI) mRNA were detected, although the level of expression was still much less than alpha 1(VI) and alpha 2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha 1(VI) and alpha 2(VI), no stable collagen VI protein was produced without expression of alpha 3(VI). The alpha 1(VI) and alpha 2(VI) chains produced in the absence of alpha 3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha 3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha 3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha 3(VI) N6-C5 chains associated with endogenous alpha 1(VI) and alpha 2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha 3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The collagens, a large protein family, form highly organized supramolecular assemblies in the extracellular space surrounding connective tissue cells. Although many collagen types form well characterized fibrils assembled by lateral aggregation of cross-linked collagen molecules, collagen VI is unique in forming an abundant microfibrillar extracellular network in virtually all connective tissues (see Refs. 1-4 for reviews). Collagen VI has both cell adhesion properties and a diverse range of extracellular matrix protein binding activities, suggesting a critical bridging role between the cell and the pericellular matrix. Thus, although it is likely that collagen VI provides both structural integrity and key matrix regulatory signals, the precise role of collagen VI during extracellular matrix development and remodeling has not been elucidated.

Collagen VI also differs from other collagens in that it is not secreted as a triple-helical monomer, but assembles intracellularly into antiparallel, overlapping dimers, which then align in a parallel manner to form tetramers that are secreted and aggregate to form extracellular microfibrils (5, 6). The component alpha -chains of collagen VI (alpha 1(VI), alpha 2(VI), and alpha 3(VI)) are complex multidomain polypeptides comprising short triple helical domains and large globular N- and C-terminal regions containing subdomains with sequence similarities to von Willebrand factor type A domains, fibronectin type III domains, Kunitz-type protease inhibitors, and several other recognized protein motifs (7-9). The alpha 3(VI) chain is much larger than the homologous alpha 1(VI) and alpha 2(VI) chains with an extended N-terminal globular domain (7), and multiple alternatively spliced forms of the alpha 3(VI) and alpha 2(VI) mRNAs give rise to protein variants, which have been proposed to modulate the organization of the microfilaments and interactions with cells and other matrix molecules (10-12).

Collagen VI alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains are commonly isolated from tissues in near equimolar amounts (3, 13-15) and the corresponding mRNAs are present in a 1:1:1 ratio in human skin fibroblast cultures (16, 17) leading to the conclusion that the collagen VI triple-helical molecule is a heterotrimer containing three genetically distinct chains. The collagen VI mRNAs can, however, be differentially regulated. Transforming growth factor-beta and gamma -interferon up-regulate and down-regulate alpha 3(VI) mRNA, respectively (18, 19), and both alpha 1(VI) and alpha 2(VI) mRNAs are up-regulated when fibroblasts are grown in a collagen gel (17). Likewise, alpha 1(VI) and alpha 2(VI) mRNAs are more abundant than alpha 3(VI) mRNA in corneal fibroblasts (14), in fetal skin (20), and in fetal skin fibroblasts (20). Although sequence data suggest that collagen VI monomers composed of all three chains are required to allow the formation of stable higher order structures (21), the possibility that these variations at the mRNA level may be reflected in the formation of alternative collagen VI isoforms with potentially different functions remains controversial. Assemblies apparently composed of only alpha 1(VI) and alpha 2(VI) chains (20), or a predominance of alpha 3(VI) chains (22) have been reported. It is important to determine the possible chain assembly isoforms to understand not only the normal biological roles of collagen VI, but also the affects of altered expression of collagen VI chains in diseases. For example, it has been proposed that triplication of the COL6A1 and COL6A2 genes and a subsequent stoichiometric disturbance of collagen VI chain composition may be responsible for some of the extracellular matrix manifestations of trisomy 21 (23, 24).

One way to address these questions directly and determine the role of the alpha 3(VI) in regulating intracellular collagen VI assembly would be to examine assembly of alpha 1(VI) and alpha 2(VI) in the absence of alpha 3(VI) chain expression. A mammalian cell line expressing only alpha 1(VI) and alpha 2(VI) would also be an important tool for assessing the role of helical and N- and C-terminal globular alpha 3(VI) domains in molecular assembly, microfibril formation, and architecture, and in cell adhesion and interactions with other matrix molecules, by site-directed mutagenesis and stable expression. Ideally, to allow questions of collagen VI matrix deposition and interactions to be examined, the cell model system should be competent to make collagen VI microfibrils and capable of forming an extensive cross-linked fibrillar collagen matrix in vitro containing a "tissue equivalent" composition of other extracellular matrix components. These characteristics have not been identified in a cell line to date (25).

In this study, we directly demonstrate the critical role of the alpha 3(VI) chain in collagen VI assembly by characterization of the human SaOS-2 bone cell line, which is deficient in alpha 3(VI) chain expression and produces no triple-helical collagen VI. However, the alpha 1(VI) and alpha 2(VI) chains are abundantly expressed by SaOS-2, and we demonstrate that when low level expression of the endogenous alpha 3(VI) is induced during long term culture, alpha 3(VI) synthesis rescues the alpha 1(VI) and alpha 2(VI) chains from degradation and allows secretion and deposition of collagen VI microfibrils within the fibrillar collagen matrix produced by these cells (26). To explore the alpha 3(VI) domains important in molecular assembly, SaOS-2 cells were stably transfected with an alpha 3(VI) cDNA construct, which lacked 4 of the 10 N-terminal type A subdomains. Despite the deletion, the alpha 3(VI) N6-C5 chains retained the ability to associate with endogenous alpha 1(VI) and alpha 2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These studies provide direct evidence that alpha 1(VI) and alpha 2(VI) chains do not form stable secreted assemblies and that alpha 3(VI) expression is essential for the formation of functional collagen VI molecules, and demonstrate that alpha 3(VI) subdomains N10-N7 are not required for molecular assembly.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Production of an alpha 3(VI) cDNA Expression Construct-- The alpha 3(VI) expression construct was prepared by ligating previously characterized partial cDNA clones (7). The 3' end of the alpha 3(VI) cDNA was excised from clone FM22 by digesting with PflMI, which cuts within the C2 domain, and SalI, which cleaves within the polylinker of the Bluescript II vector (Stratagene). This 2.5-kilobase fragment was ligated into the corresponding sites of F376 to generate a cDNA encoding protein domains N2-C5. A SacII fragment of this clone was excised using sites within the triple helix and the Bluescript II polylinker, and ligated into the SacII-digested FO4 cDNA, producing a clone encoding domains N8-C5. A fragment coding for domains N6-C5 was excised from the N8-C5 clone by digesting with NheI, which cuts within the N7 domain, filling in the restriction site overhang with the Klenow fragment of DNA polymerase I, followed by cleavage at the 3' NotI polylinker site. This fragment was ligated into the expression plasmid pRc/AC (27), which had also been cleaved with NheI, made blunt-ended with Klenow DNA polymerase I, and then digested with NotI. The resulting expression construct, alpha 3(VI) N6-C5, encoded the BM-40 signal sequence, 54 amino acids of the alpha 3(VI) N7 domain, domains N6-C5, and the 3' untranslated region and polyadenylation sequence, in the vector pRc/CMV (Invitrogen), which also encodes the neomycin phosphotransferase gene conferring resistance to the antibiotic G418.

Cell Culture and Transfection-- A human osteosarcoma (SaOS-2) cell line (28, 29) (ATCC HTB-85), mouse NIH/3T3 (ATCC CRL-1658) and 3T6 fibroblasts (ATCC CCL-96), and a rat osteogenic sarcoma (UMR 106) cell line (30) (ATCC CRL-1661) were obtained from American Type Culture Collection. Mouse Mov-13 cells (31) were provided by Dr. R. Jaenisch (Whitehead Institute for Biomedical Research, Cambridge, MA). A mouse osteoblastic cell line, MC-3T3 E1 (32) was obtained from Dr. H. Kodama (Tohoku Dental College, Kohriyama, Japan). Human dermal fibroblasts were established from skin biopsies (33). Cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM)1 containing 10% (v/v) fetal calf serum as described previously (33). From confluence (day 0), the cell cultures were grown in DMEM containing 10% (v/v) fetal calf serum, and supplemented daily with sodium ascorbate to a final concentration of 0.25 mM (34). SaOS-2 cells were transfected with the alpha 3(VI) N6-C5 cDNA expression construct using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's protocol. Stably transfected cells were selected in growth medium containing 500 µg/ml G418 (Life Technologies, Inc.). Individual G418-resistant colonies were isolated and expanded into cell lines. G418 was removed from the culture medium after the fourth passage.

Northern Blot Analysis-- Total RNA was prepared by the guanidine hydrochloride extraction method of Wake and Mercer (35). RNA samples were analyzed under denaturing conditions on 0.8% (w/v) agarose gels and transferred to nitrocellulose or nylon filters (36). Filters were hybridized to [alpha -32P]dCTP-labeled alpha 1(VI), alpha 2(VI), and alpha 3(VI) cDNA probes (P18, P1, and P24; Ref. 21), washed and specific hybridization visualized by autoradiography (36).

Antibodies-- Antibodies specifically recognizing collagen VI alpha 1(VI) (37) and alpha 3(VI) chains (38) have been described previously. Antibodies used for immunoprecipitations were raised against pepsin-digested collagen VI and, although they both recognized all three collagen VI subunits on Western blots, one precipitated only assemblies of alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains (Life Technologies, Inc.), and the other precipitated both assembled and individual, unassociated alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains (39).

Collagen Biosynthetic Labeling-- Skin fibroblasts and SaOS-2 cells in 10-cm2 culture dishes were grown to confluence, supplemented with 0.25 mM sodium ascorbate for 24 h, then biosynthetically labeled for 18 h with 100 µCi/ml [35S]methionine (Tran35S-labelTM 1032 Ci/mmol, ICN Pharmaceuticals, Inc.) in methionine-free and serum-free DMEM containing 0.25 mM sodium ascorbate. The medium was removed and added to an equal volume of 2× NET buffer (NET buffer is 50 mM Tris-HCl, pH 7.5 containing 1 mM EDTA, 150 mM NaCl, and 0.1% Nonidet P-40). Protease inhibitors were included at the following final concentrations: 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM N-ethylmaleimide (NEM), and 5 mM EDTA. The cell layer was solubilized in 50 mM Tris/HCl, pH 7.5 containing 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF and 20 mM NEM. Cell lysates and medium samples were clarified by centrifugation and precleared with 30 µl of anti-fibronectin antibody (Life Technologies, Inc.) and 100 µl of 20% Protein A-Sepharose (Amersham Pharmacia Biotech) at 4 °C for 2 h. Supernatants were incubated with 5 µl of collagen VI antiserum (Life Technologies, Inc.) and 100 µl of 20% Protein A-Sepharose for 18 h at 4 °C, and the immunoprecipitated complexes washed two times for 30 min each with fresh NET buffer, then once with 10 mM Tris-HCl, pH 7.5, containing 0.1% Nonidet P-40.

For pulse-chase experiments, confluent SaOS-2 cells were incubated overnight in medium containing 10% fetal calf serum and 0.25 mM sodium ascorbate, preincubated for 30 min in methionine-free and serum-free DMEM supplemented with 0.25 mM sodium ascorbate, then pulse-labeled for 1 h with 100 µCi/ml [35S]methionine. The labeling medium was removed, after which the cells were washed with serum-free DMEM containing 50 mM cold methionine and 0.25 mM sodium ascorbate, and then incubated in this chase medium for up to 24 h. Medium and cell layer fractions were harvested as above and the collagen VI immunoprecipitated with 5 µl of the collagen VI antiserum, which precipitates both assembled and unassociated alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains (39).

Pepsin Digestion-- Immunoprecipitated collagen VI was resuspended in 0.5 M acetic acid containing 100 µg/ml pepsin and incubated at 4 °C for 16 h. Digestion was terminated by lyophilization.

Extraction of Collagen VI from the in Vitro Accumulated Extracellular Matrix-- At the appropriate time points, the matrix was harvested by rinsing the cell layer twice with phosphate-buffered saline and scraping it into 20 mM sodium phosphate buffer, pH 7.4 containing fresh protease inhibitors (20 mM NEM, 1 mM PMSF, 25 mM EDTA). The insoluble matrix was collected by centrifugation (10,000 × g, 10 min) and rinsed briefly in cold distilled water containing protease inhibitors. The pellet was solubilized in 50 mM Tris-HCl, pH 7.5 containing 0.5% (w/v) SDS and 8 M urea by heating at 80 °C for 5 min.

SDS-PAGE and Immunoblotting-- Collagen VI chains were analyzed by SDS-PAGE on 5% (w/v) polyacrylamide gels and pepsinized type VI collagen on 7.5% (w/v) polyacrylamide gels along with [14C]methylated protein molecular weight markers (Amersham Pharmacia Biotech). Where indicated, samples were reduced before electrophoresis by the addition of dithiothreitol to a final concentration of 25 mM. Collagen VI dimers and tetramers were analyzed on 2.4% (w/v) acrylamide/0.5% (w/v) agarose composite gels (40) under non-reducing conditions. Molecular size standards were the reduced (230 kDa and 400 kDa) and unreduced (900 kDa) forms of laminin (Boehringer Mannheim). Radioactively labeled proteins were detected by fluorography (33, 41) or imaged using a PhosphorImager (Molecular Dynamics, STORMTM).

For immunoblotting, collagen chains resolved by SDS-PAGE were electrophoretically transferred to nitrocellulose filters. Bound antibodies were detected using horseradish peroxidase-conjugated Protein A (Bio-Rad) and an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).

Indirect Immunofluorescence-- SaOS-2 cells were grown in eight-well chamber glass slides (Nunc Inc.) under culture conditions described above, washed with phosphate-buffered saline, fixed with ice-cold methanol for 10 min, then air-dried. Slides were incubated with either primary collagen VI antibody (Life Technologies, Inc.) or non-immune rabbit serum for 1 h at room temperature and bound antibody detected using fluorescein-conjugated AffiniPure donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories, Inc.). Slides were mounted in FluorSaveTM reagent (Calbiochem) then viewed and photographed using a Zeiss fluorescence microscope.

    RESULTS
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Procedures
Results
Discussion
References

Collagen VI mRNA Expression in Cell Lines-- A range of cell lines capable of producing type I collagen and other extracellular matrix components were screened for collagen VI mRNA expression by Northern blotting. Two cell lines, NIH/3T3 and MC-3T3 E1, produced trace quantities of alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNAs; Mov13 and 3T6 synthesized trace amounts of alpha 1(VI) and alpha 2(VI) mRNA; and UMR 106 did not produce detectable levels of any collagen VI subunit mRNA (data not shown). Primary cultures of dermal fibroblasts, however, expressed abundant alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNA (Fig. 1, b, d, and f, lane 1), reflecting the high level of expression of collagen VI in skin (16). Confluent SaOS-2 cells also produced alpha 1(VI) and alpha 2(VI) mRNA, at levels comparable to that of skin fibroblasts, but were totally deficient in alpha 3(VI) transcription (Fig. 1f, lane 2) (14). The SaOS-2 cell line was thus the only cell line examined that expressed alpha 1(VI) and alpha 2(VI) mRNAs at relatively high levels, making it a potentially valuable tool for the analysis of collagen VI assembly in the absence of alpha 3(VI), and subsequent stable transfection and expression of alpha 3(VI) chains.


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Fig. 1.   Expression of alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNAs in SaOS-2 cells during long term culture with ascorbate. SaOS-2 cells were maintained for up to 30 days after confluence in the presence of 0.25 mM sodium ascorbate. Lane 1 contains 5 µg of positive control total RNA isolated from skin fibroblasts. Lanes 2-8 contain 10 µg of SaOS-2 RNA harvested at 0, 2, 3, 10, 12, 15, and 30 days, respectively. Denaturing agarose gels were stained with ethidium bromide (a, c, and e), the RNA was transferred to nylon filters then hybridized to [alpha -32P]dCTP-labeled alpha 1(VI) (b), alpha 2(VI) (d), and alpha 3(VI) (f and g) cDNA probes. Panels b, d, and f show overnight exposures, whereas panel g shows a 2-week exposure of the alpha 3(VI)-probed filter. Migration positions of the alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNA bands and of the 28 S and 18 S rRNAs are indicated.

SaOS-2 Collagen VI mRNA Expression during Long Term Culture with Ascorbic Acid-- SaOS-2 bone cells can be induced to lay down an extensive type I collagen matrix, which can mineralize in vitro by extended culture in the presence of sodium ascorbate (26). Ascorbate is an essential cofactor for collagen hydroxylation and subsequent helix formation and secretion, and this technique has been used extensively to stimulate collagen matrix deposition and generate in vitro "tissue equivalent" extracellular matrices (34, 42). Because during this ascorbate-induced matrix formation in vitro, major changes in collagen gene expression have been reported (34), it was of importance to see if alpha 3(VI) gene expression was influenced by long term culture and type I collagen matrix deposition. The changes in mRNA expression of the three alpha -chains over a 30-day culture period with sodium ascorbate is shown in Fig. 1. Steady-state levels of alpha 1(VI) mRNA and alpha 2(VI) mRNA remained constant over the first 10-12 days of culture (Fig. 1, b and d, lanes 2-6), but diminished significantly between days 15 and 30 (lanes 7 and 8). Although the steady state levels of alpha 3(VI) mRNA remained below the level of detection under identical analytical conditions during the entire 30-day culture period (Fig. 1f), overexposure of the Northern blots demonstrated that low level alpha 3(VI) expression is gradually induced during the extended culture period (Fig. 1g), with the highest levels of expression at days 15-30 (Fig. 1g, lanes 7 and 8).

Collagen VI Matrix Deposition in SaOS-2 Cultures-- Collagen VI matrix deposition during the 30-day culture period was assessed by immunoblotting of urea-extracted matrix using chain-specific antibodies (Fig. 2). Skin fibroblasts, which have steady state levels of alpha 1(VI) and alpha 2(VI) mRNA similar to those in the SaOS-2 cells, produced an extensive collagen VI matrix by 8 days of culture (Fig. 2, a-c, lane 1). In contrast, alpha 1(VI) and alpha 2(VI) chains were not detected in the SaOS-2 extracellular matrix until day 15 (Fig. 2, b and c, lanes 2-6), despite the abundant synthesis of alpha 1(VI) and alpha 2(VI) mRNA. However, from day 15, which coincided with the induction of expression of alpha 3(VI) mRNA, the SaOS-2 cells did produce a small amount of collagen VI matrix, which was detected at day 15 with just the alpha 1(VI) antibody (Fig. 2c, lanes 7), but by day 30 was detected by both the alpha 1(VI) and alpha 3(VI) antibodies (Fig. 2, b and c, lane 8). The appearance of collagen VI chains in the matrix only after low level expression of alpha 3(VI) mRNA, suggested that the alpha 1(VI) and alpha 2(VI) chains were unable to associate into collagen VI microfibrils without the co-expression of alpha 3(VI).


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Fig. 2.   Immunoblot analysis of collagen VI accumulated within a SaOS-2 extracellular matrix. The matrix produced by SaOS-2 cells grown for up to 30 days in the presence of 0.25 mM sodium ascorbate was solubilized in 8 M urea, resolved by 5% SDS-PAGE under reducing conditions, transferred onto nitrocellulose, and analyzed by immunoblotting. Lane 1, skin fibroblast matrix harvested at day 8; lanes 2-8, SaOS-2 cell layers harvested at 0, 2, 3, 10, 12, 15, and 30 days, respectively. Lanes 2-8 contain 3 times more matrix extract than lane 1. Filters were probed with antibodies specifically recognizing either alpha 3(VI) (a and b) or alpha 1(VI) chains (c) and bound antibody detected by chemiluminescence. Panel a shows a shorter exposure of lane 1 in panel b. The migration positions of the alpha 1(VI) and alpha 3(VI) chains are indicated.

Fate of the alpha 1(VI) and alpha 2(VI) Polypeptides Synthesized by SaOS-2-- The demonstration of alpha 1(VI) and alpha 2(VI) mRNA and the inability to detect collagen VI matrix formation in the absence of the alpha 3(VI) provided strong evidence that the alpha 3(VI) chain was essential for molecular assembly and matrix formation. To determine if the alpha 1(VI) and alpha 2(VI) chains were retained within the cell and degraded, or were secreted as individual polypeptide chains or as stable triple helical assemblies, collagen VI chains synthesized during a 1-h pulse-labeling were chased for up to 24 h, immunoprecipitated, and examined by both reducing and non-reducing electrophoresis. Immediately after the pulse-labeling alpha 1(VI) and alpha 2(VI) were abundant intracellularly (Fig. 3a, lane 1), and a proportion of these chains had associated into higher order disulfide-bonded structures (Fig. 3b, lane 1). These assemblies migrated with the molecular weight expected of alpha -chain dimers and most likely represent disulfide-bonded alpha 1(VI) and alpha 2(VI) homo- or heterodimers. Because alpha 3(VI) mRNA is not detectable in SaOS-2 cells at confluence (Fig. 1f, lane 2), the feint band migrating above alpha 1(VI) and alpha 2(VI) is most probably fibronectin, which has not been completely removed before immunoprecipitation with the collagen VI antibody (20). The intracellular alpha 1(VI) and alpha 2(VI) diminished gradually during the 24-h chase period (Fig. 3a, lanes 2-6) with the concomitant appearance of a small proportion of unassociated chains and disulfide-bonded alpha 1(VI) and alpha 2(VI) dimers in the medium (Fig. 3, c and d). However, when the amount of total intracellular and secreted collagen VI remaining at 24 h was compared with that synthesized during the 1-h labeling, it was clear that the alpha (VI) and alpha 2(VI) chains were not efficiently secreted and the vast majority were retained intracellularly and degraded. When the SaOS-2 intracellular and extracellular alpha 1(VI) and alpha 2(VI) chains were digested with pepsin, they were completely degraded, clearly demonstrating that these chains have not formed helical assemblies (data not shown).


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Fig. 3.   Pulse/chase analysis of collagen VI biosynthesis in SaOS-2 cells. Confluent SaOS-2 cells were supplemented with 0.25 mM sodium ascorbate for 24 h, pulse-labeled with [35S]methionine for 1 h, then chased for up to 24 h in medium containing 50 mM cold methionine. After preclearing of the samples with an anti-fibronectin antibody, collagen VI was immunoprecipitated using an antibody that precipitates both triple helical molecules and free polypeptide chains (39), then resolved on 5% polyacrylamide gels and detected by fluorography. Lanes 1-6, cell layer (a and b) and medium (c and d) samples harvested at 0, 1, 3, 6, 12, and 24 h of chase, respectively, run under either reducing (a and c) or non-reducing (b and d) conditions. The migration positions of the alpha 1(VI) and alpha 2(VI) chains, and fibronectin (FN) are indicated. 1 denotes the migration position of disulfide-bonded dimers of alpha 1(VI) and alpha 2(VI) chains.

Stable Expression of the alpha 3(VI) N6-C5 Construct in SaOS-2 Cells-- To confirm the essential role of the alpha 3(VI) chain in collagen VI assembly and secretion and explore the importance of the alternatively spliced N-terminal domains in microfibril formation, SaOS-2 cells were transfected with the alpha 3(VI) N6-C5 expression construct (Fig. 4). Individual, stably transfected clones were selected in medium containing G418 and then screened for expression of alpha 3(VI) mRNA by Northern blot. Two cell lines expressing significant amounts of alpha 3(VI) mRNA as well as endogenous alpha 1(VI) and alpha 2(VI) mRNAs were obtained (data not shown). The cell line expressing the highest level of alpha 3(VI) mRNA, at levels approximately equal to alpha 1(VI) and alpha 2(VI) mRNAs, was used in subsequent experiments.


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Fig. 4.   Schematic diagram of the collagen VI subunits. Collagen VI alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains including their various C- and N-terminal protein domains and the triple helix (black box) are shown in a. The shaded type A modules, alpha 3(VI) N10, N9, and N7, and alpha 2(VI) C2, have been shown to undergo alternative splicing. The alpha 3(VI) protein domains included in the expression construct N6-C5 are illustrated in b.

The alpha 3(VI) N6-C5 Chain Rescues the Endogenous alpha 1(VI) and alpha 2(VI) and Restores Collagen VI Assembly and Secretion-- To determine if the alpha 3(VI) N6-C5 chain was able to associate with endogenous alpha 1(VI) and alpha 2(VI), cells were metabolically labeled for 18 h with [35S]methionine, and the collagen VI in the cell and medium immunoprecipitated with an antibody that precipitates only collagen VI assemblies and not the unassociated polypeptide chains (Life Technologies, Inc.). No collagen VI was immunoprecipitated from either the cell or medium fraction of untransfected SaOS-2 cells (Fig. 5, lanes 2 and 3), confirming that in the absence of the alpha 3(VI), alpha 1(VI), and alpha 2(VI) chains did not form triple helical collagen VI molecules. In contrast, the alpha 3(VI) N6-C5 chain produced by the transfected SaOS-2 cells associated with the endogenous alpha 1(VI) and alpha 2(VI), rescued them from intracellular degradation, and formed collagen VI assemblies which were efficiently secreted (Fig. 5, lanes 4 and 5).


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Fig. 5.   Electrophoretic analysis of immunoprecipitated collagen VI. Untransfected SaOS-2 cells and cells transfected with the alpha 3(VI) N6-C5 construct were biosynthetically labeled with [35S]methionine for 18 h. Collagen VI in the cell layer (C) and medium (M) fractions was immunoprecipitated with an antibody capable of precipitating collagen VI assemblies but not free chains (Life Technologies, Inc.), and resolved on a 5% (w/v) polyacrylamide gel under reducing conditions. Collagen VI alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains are indicated. The size in kDa of the [14C]methylated protein molecular size standards (lane 1) are shown on the left.

Collagen VI Molecules Containing N-terminally Deleted alpha 3(VI) Chains Form Disulfide-bonded Tetramers-- The normal pathway of collagen VI assembly involves the intracellular assembly of triple helical monomers (3 chains) to form disulfide-bonded dimers (6 chains), and then tetramers (12 chains). To determine if N-terminal alpha 3(VI) domains are required for this higher order assembly, the collagen VI synthesized by SaOS-2 cells transfected with alpha 3(VI) N6-C5 was analyzed on non-reducing acrylamide/agarose composite gels. The predominant form of collagen VI secreted by human fibroblasts migrated with a molecular mass of about 2,000 kDa, as expected for collagen VI tetramers (Fig. 6, lane 4). A small proportion of the collagen VI was secreted as dimers, and a band migrating at around 4,000 kDa suggested that some tetramers had formed end-to-end associations (Fig. 6, lane 4). The same pattern of predominantly collagen VI tetramers with a small proportion of dimers and some higher order assembly, was seen in the medium of the transfected SaOS-2 cells (Fig. 6, lane 2), clearly demonstrating that alpha 3(VI) domains N10-N7 are not required for intracellular monomer, dimer, or tetramer assembly. Rescue of the endogenous alpha 1(VI) and alpha 2(VI) chains by alpha 3(VI) N6-C5, and the efficient secretion of collagen VI tetramers, strongly suggested that these chains had folded to form a stable triple helix. This was directly demonstrated by the presence of pepsin-resistant forms of collagen VI in both the cell and medium of SaOS-2 cells transfected with the alpha 3(VI) N6-C5 construct (data not shown).


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Fig. 6.   Analysis of collagen VI dimer and tetramer assembly by composite gel electrophoresis. Human fibroblasts (control) and SaOS-2 cells transfected with the alpha 3(VI) N6-C5 construct were labeled with [35S]methionine for 18 h and the collagen VI immunoprecipitated from the cell layer (C) and medium (M) resolved on a 2.4% acrylamide, 0.5% agarose gel under non-reducing conditions. The migration position of disulfide-bonded collagen VI dimers (6 chains), tetramers (12 chains), and non-covalently associated double tetramers ((tet)2) are indicated. The migration and size in kDa of the reduced and unreduced forms of laminin are shown on the left.

alpha 3(VI) Domains N10-N7 Are Not Required for Assembly of Collagen VI Microfibrils-- To determine if collagen VI tetramers containing the alpha 3(VI) N6-C5 chain were incorporated into a microfibrillar matrix, SaOS-2 cell cultures were grown in the presence of sodium ascorbate, stained with a collagen VI antibody and examined by immunofluorescence microscopy. By day 12 of culture, untransfected SaOS-2 cells had deposited only minute amounts of collagen VI into their extracellular matrix (Fig. 7a) consistent with the low level of alpha 3(VI) mRNA expression detected at this time (Fig. 1g). In contrast, SaOS-2 cells expressing alpha 3(VI) N6-C5 chains had deposited an extensive collagen VI network (Fig. 7b) indicating that this shortened alpha 3(VI) chain contained all the domains required, not only for monomer, dimer, and tetramer assembly, but also for the end-to-end association of tetramers to form matrix microfibrils.


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Fig. 7.   Collagen VI in the in vitro accumulated extracellular matrix. Untransfected SaOS-2 cells (a) and SaOS-2 cells transfected with the alpha 3(VI) N6-C5 construct (b) were grown for 12 days after confluence in the presence of 0.25 mM sodium ascorbate, then fixed and incubated with a collagen VI antibody (Life Technologies, Inc.). Bound antibody was detected using fluorescein-conjugated donkey anti-rabbit IgG.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The experiments presented here provide the first direct evidence for the crucial role of the alpha 3(VI) chain in collagen VI molecular assembly and matrix formation and demonstrate that the functional collagen VI triple-helical monomer is a heterotrimer which contains alpha 3(VI) chains as well as alpha 1(VI) and alpha 2(VI), most probably in the ratio of 1:1:1. Our data demonstrate that the human osteosarcoma cell line, SaOS-2 does not synthesize alpha 3(VI) in short term culture, but produces abundant alpha 1(VI) and alpha 2(VI), which are unable to form helical assemblies and are unstable. Furthermore, long term culture studies where the synthesis of alpha 3(VI) was induced, demonstrating that the cells have all the necessary cellular machinery to correctly assemble and secrete collagen molecules composed of alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains, and deposit collagen VI into the extracellular matrix.

These data showing the central role of the alpha 3(VI) chain in allowing stable protein formation support previous predictions from sequence analyses that collagen VI molecules composed of all three chains are more stable than other assembly alternatives (21), and are consistent with studies demonstrating that when a specific reduction in the steady-state level of alpha 3(VI) mRNA, but not alpha 1(VI) or alpha 2(VI) mRNA is induced by gamma -interferon treatment of skin fibroblasts, all three collagen VI subunits are secreted in reduced amounts (19). Furthermore, the deposition of collagen VI in the developing mouse extracellular matrix also correlates with expression of the alpha 3(VI) gene, irrespective of the earlier expression of the alpha 1(VI) and alpha 2(VI) chains (43).

Variations in the chain composition of triple-helical collagen VI molecules have, however, been described. In the most comprehensive study (20), post-confluent fetal bovine skin fibroblasts secreted pepsin-resistant collagen VI molecules apparently comprised predominately of alpha 1(VI) and alpha 2(VI) chains. Fetal skin extracts also contained an excess of alpha 1(VI) and alpha 2(VI) chains (20). Difficulties in the interpretation of these experiments arise from the variable nature of pepsin digestion and the sensitivity of the alpha 3(VI) globular domains to proteolytic degradation, both of which tend to give the impression of an overabundance of alpha 1(VI) and alpha 2(VI) chains. Collagen VI alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains are each degraded to different sized polypeptides by pepsin, and further heterogeneity can arise by non-uniform cleavage within each chain (3). We commonly see only two bands after pepsin digestion of collagen VI immunoprecipitated from human fibroblast cultures, even though the alpha 3(VI) chain is a major component before pepsin digestion.2 These bands show gel migrations consistent with the major pepsin-resistant forms of the alpha 1(VI) and alpha 2(VI) chains. This phenomenon has also been observed when examining collagen VI extracted from nucleus pulposis. Native collagen VI from this tissue shows a 1:1:1 ratio of the three chains, but the alpha 3(VI) chain is apparently much less abundant after pepsin digestion (40). It seems likely, then, that variable pepsin digestion commonly results in forms of alpha 3(VI) that comigrate with pepsin-resistant alpha 1(VI) and/or alpha 2(VI). In addition, we find that the globular domains of native alpha 3(VI) chains are readily removed by proteases present in tissue culture samples, resulting in the recovery of only 140-kDa components, and in fact, many early studies on "intact" collagen VI failed to detect the large >200-kDa form of the alpha 3(VI) chain because of its lability (3). To prevent this proteolysis and allow recovery of intact alpha 3(VI) chains, samples must be analyzed rapidly after extraction in the presence of fresh protease inhibitors, and not stored before purification, even for short periods.2 Examination of collagen VI biosynthesis and assembly in SaOS-2 cells overcomes these problems of variable proteolysis, which make it difficult to accurately determine the molecular formula of collagen VI in both intact and pepsin-digested samples, and demonstrated directly that no triple-helical collagen VI assemblies were produced when the alpha 3(VI) chain was not expressed. This situation is analogous to that of collagen IX where, in mice, inactivation of the Col9a1 gene, which encodes the alpha 1(IX) chain, leads to a functional knock-out of all the collagen IX chains, even though mRNAs for alpha 2(IX) and alpha 3(IX) are transcribed normally (44). The alpha 1(IX) chain thus plays a pivotal role in the assembly of stable triple-helical collagen IX, comparable to the central role of the alpha 3(VI) in collagen VI assembly demonstrated in this study.

In the absence of alpha 3(VI), the alpha 1(VI) and alpha 2(VI) chains produced by SaOS-2 cells are largely retained within the cell as unassembled polypeptides and are degraded. In contrast, when transfected with either alpha 1(VI) or alpha 2(VI) cDNAs, 293 cells efficiently secreted these chains as non-helical polypeptide monomers or homodimers (37), indicating that cells which do not produce endogenous collagen VI may lack the quality control mechanisms present in SaOS-2 that normally prevent secretion of unassembled chains (25). The mechanism of intracellular degradation of the unassembled alpha 1(VI) and alpha 2(VI) was not determined, but for other unassembled normal collagen species, such as the proalpha 2(I) chain in Mov-13 cells and human fibroblasts, this degradation occurs predominantly via a post-endoplasmic reticulum non-lysosomal mechanism (45), similar to that described for basal collagen degradation (46, 47). In contrast, structurally abnormal malfolded collagens appear to be rapidly degraded by endoplasmic reticulum-mediated quality control processes (45). A small proportion of the alpha 1(VI) and alpha 2(VI) were secreted as monomeric peptides or disulfidebonded dimers, but these were pepsin-sensitive, thus not in a stable helical configuration and unable to play a structural role in the matrix. Intact collagen VI microfibrils are susceptible to degradation by serine proteinases and matrix metalloproteinase-9 (MMP-9) (48), and collagen VI assemblies lacking disulfide bonds are digested by MMP-2 (49). Secreted non-helical, non-disulfide-bonded chains could be expected to be even more susceptible to proteolytic attack (3) and are unlikely to persist in the extracellular matrix.

This finding that alpha 1(VI) and alpha 2(VI) do not form stable collagen VI assemblies has important implications for the interpretation of studies examining collagen VI expression and regulation during development, in response to growth factors, and in disease. Although we cannot formally exclude the possibility that stable collagen VI isoforms containing only alpha 1(VI) and alpha 2(VI) can form under some conditions, our data indicate that coordinated temporal and spatial expression of the three alpha -chains will be necessary for collagen VI matrix deposition during development, and that increased mRNA levels will not necessarily be reflected in the amount of functional secreted collagen VI. For example, the COL6A1 and COL6A2 genes, encoding the alpha 1(VI) and alpha 2(VI) subunits, are both found on chromosome 21, and some studies have suggested that alterations in the extracellular matrix of trisomy 21 skin could be explained by elevated levels of collagen VI or a stoichiometric disturbance in the chain composition of collagen VI (23, 24). However, excess alpha 1(VI) and alpha 2(VI) chains in trisomy 21 cells would most likely not form stable molecules and be degraded before secretion. Likewise, increased accumulation of collagen VI in the extracellular matrix of these patients would only result if alpha 3(VI) chains were normally synthesized in excess and collagen VI assembly was limited by the availability of alpha 1(VI) and alpha 2(VI).

The SaOS-2 cell line is also a powerful model system for stable transfection and expression of alpha 3(VI) chains modified by site-directed mutagenesis, and will facilitate studies designed to define the roles of alpha 3(VI) domains in molecular assembly, microfibril formation and architecture, cell adhesion, and interactions with other matrix molecules. The potential of this approach was demonstrated here by the establishment of SaOS-2 cell lines stably expressing the transfected alpha 3(VI) N6-C5 cDNA at high levels. In transfected cells, alpha 3(VI) chains restored normal collagen VI biosynthesis by associating with endogenous alpha 1(VI) and alpha 2(VI), rescuing them from intracellular degradation, and forming collagen VI dimers and tetramers which were secreted and deposited into the extracellular matrix.

Our experiments also address the unsolved issue of the role of the alpha 3(VI) extended N-terminal domain in collagen VI assembly and function. The alpha 3(VI) construct used in this study did not contain all the N-terminal repetitive subdomains, but lacked subdomains N10-N7. These deleted alpha 3(VI) chains retained the ability to assemble intracellularly into heterotrimeric monomers, and higher order oligomers. The role of specific motifs in driving assembly is not known, but by analogy to other collagen types it might be expected that the C-terminal globular domains would play a fundamental role in chain selection, and initiating helix formation (50, 51). Normal intracellular assembly of the alpha 3(VI) N6-C5 chains is also consistent with rotary shadowing studies, which have suggested that dimers are stabilized by interactions between the C-terminal globular domains of one monomer and the triple helix of the adjacent molecule (52, 53), whereas the important interactions for tetramer stability are between the N-terminal regions of the triple helix (52). The formation of matrix microfibrils involves the end-to-end association of tetramers, with an overlap of the terminal regions of the tetramers bringing the N- and C-terminal globular domains into close contact (4, 52, 53). Although the exact nature of the forces stabilizing collagen VI microfibrils have not been identified, interactions between the N- and C-terminal globular domains, which have a high affinity for each other, are thought to be critical (53). The ability of collagen VI tetramers containing alpha 3(VI) N6-C5 chains to form microfibrils clearly demonstrates that subdomains N10-N7 are not required for either intracellular or extracellular collagen VI assembly.

The N-terminal subdomains of the alpha 3(VI) chain, N10-N2, are each encoded by a separate exon (10, 11, 54). Three of these exons, encoding subdomains N10, N9, and N7, are known to be alternatively spliced in both human and chicken, predicting multiple alpha 3(VI) chains, which may lack one to three subdomains (10, 11, 54). Interestingly, recent studies characterizing alpha 3(VI) splice variants in the mouse have demonstrated splicing of additional exons, including exons encoding subdomains N8 and N5.3 One of the relatively abundant isoforms found was an alpha 3(VI) N6-C5 chain, and our data show that this chain assembles normally and is incorporated into the extracellular matrix. The functional significance of the alpha 3(VI) splicing events is not known; however, as the alpha 3(VI) N-terminal region has been shown to bind in vitro to type I collagen (55) and heparin (38), and subdomains N10-N7 are not required for assembly, it seems reasonable to speculate that alternative splicing may modulate collagen VI interactions with other matrix ligands and allow the formation of microfibrils with tissue-specific functions.

    FOOTNOTES

* This work was supported by grants from the National Health and Medical Research Council of Australia and the Royal Children's Hospital Research Foundation (to J. F. B.) and by National Institutes of Health Grant AR38912 (to M.-L. C.).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.

** To whom correspondence should be addressed. Tel.: 61-3-9345-6367; Fax: 61-3-9345-6668/6367; E-mail: bateman{at}cryptic.rch.unimelb.edu.au.

1 The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.

2 S. R. Lamandé and J. F. Bateman, unpublished data.

3 M. Dziadek, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Kielty, C. M., Hopkinson, I., and Grant, M. E. (1993) in Connective Tissue and Its Heritable Disorders: Molecular, Genetic, and Medical Aspects (Royce, P. M., and Steinmann, B., eds), pp. 103-147, Wiley-Liss, Inc., New York
  2. Bateman, J. F., Lamandé, S. R., and Ramshaw, J. A. M. (1996) in Extracellular Matrix (Comper, W. D., ed), pp. 22-67, Harwood Academic Publishers, Amsterdam
  3. Timpl, R., and Engel, J. (1987) in Structure and Function of Collagen Types (Mayne, R., and Burgeson, R. E., eds), pp. 105-143, Academic Press, Orlando, FL
  4. Timpl, R., and Chu, M.-L. (1994) in Extracellular Matrix Assembly and Structure (Yurchenco, P. D., Birk, D., and Mecham, R. P., eds), pp. 207-242, Academic Press, Orlando, FL
  5. Colombatti, A., Bonaldo, P., Ainger, K., Bressan, G. M., Volpin, D. (1987) J. Biol. Chem. 262, 14454-14460[Abstract/Free Full Text]
  6. Engvall, E., Hessle, H., and Klier, G. (1986) J. Cell Biol. 102, 703-710[Abstract]
  7. Chu, M. L., Zhang, R. Z., Pan, T. C., Stokes, D., Conway, D., Kuo, H. J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., Timpl, R. (1990) EMBO J. 9, 385-393[Abstract]
  8. Bonaldo, P., and Colombatti, A. (1989) J. Biol. Chem. 264, 20235-20239[Abstract/Free Full Text]
  9. Colombatti, A., and Bonaldo, P. (1997) Blood 77, 2305-2315[Medline] [Order article via Infotrieve]
  10. Stokes, D. G., Saitta, B., Timpl, R., and Chu, M. L. (1991) J. Biol. Chem. 266, 8626-8633[Abstract/Free Full Text]
  11. Doliana, R., Bonaldo, P., and Colombatti, A. (1990) J. Cell Biol. 111, 2197-2205[Abstract]
  12. Saitta, B., Stokes, D. G., Vissing, H., Timpl, R., and Chu, M. L. (1990) J. Biol. Chem. 265, 6473-6480[Abstract/Free Full Text]
  13. Jander, R., Rauterberg, J., and Glanville, R. W. (1983) Eur. J. Biochem. 133, 39-46[Abstract]
  14. Chu, M. L., Mann, K., Deutzmann, R., Pribula-Conway, D., Hsu-Chen, C. C., Bernard, M. P., Timpl, R. (1987) Eur. J. Biochem. 168, 309-317[Abstract]
  15. Schreier, T., Winterhalter, K. H., and Trueb, B. (1987) FEBS Lett. 213, 319-323[CrossRef][Medline] [Order article via Infotrieve]
  16. Olsen, D. R., Peltonen, J., Jaakkola, S., Chu, M. L., Uitto, J. (1989) J. Clin. Invest. 83, 791-795[Medline] [Order article via Infotrieve]
  17. Hatamochi, A., Aumailley, M., Mauch, C., Chu, M. L., Timpl, R., Krieg, T. (1989) J. Biol. Chem. 264, 3494-3499[Abstract/Free Full Text]
  18. Heckmann, M., Aumailley, M., Chu, M. L., Timpl, R., Krieg, T. (1992) FEBS Lett. 310, 79-82[CrossRef][Medline] [Order article via Infotrieve]
  19. Heckmann, M., Aumailley, M., Hatamochi, A., Chu, M. L., Timpl, R., Krieg, T. (1989) Eur. J. Biochem. 182, 719-726[Abstract]
  20. Kielty, C. M., Boot-Handford, R. P., Ayad, S., Shuttleworth, C. A., Grant, M. E. (1990) Biochem. J. 272, 787-795[Medline] [Order article via Infotrieve]
  21. Chu, M. L., Conway, D., Pan, T. C., Baldwin, C., Mann, K., Deutzmann, R., Timpl, R. (1988) J. Biol. Chem. 263, 18601-18606[Abstract/Free Full Text]
  22. Katagiri, K., Takasaki, S., Fujiwara, S., Kayashima, K., Ono, T., and Shinkai, H. (1996) J. Dermatol. Sci. 13, 37-48[CrossRef][Medline] [Order article via Infotrieve]
  23. Brand-Saberi, B., Floel, H., Christ, B., Schulte-Vallentin, M., and Schindler, H. (1994) Anatomischer Anzeiger 176, 539-547[Medline] [Order article via Infotrieve]
  24. Brand-Saberi, B., Epperlein, H. H., Romanos, G. E., Christ, B. (1994) Cell Tissue Res. 277, 465-475[CrossRef][Medline] [Order article via Infotrieve]
  25. Colombatti, A., Mucignat, M. T., and Bonaldo, P. (1995) J. Biol. Chem. 270, 13105-13111[Abstract/Free Full Text]
  26. McQuillan, D. J., Richardson, M. D., Bateman, J. F. (1995) Bone 16, 415-426[CrossRef][Medline] [Order article via Infotrieve]
  27. Mayer, U., Poschl, E., Nischt, R., Specks, U., Pan, T. C., Chu, M. L., Timpl, R. (1994) Eur. J. Biochem. 225, 573-580[Abstract]
  28. Fogh, J., and Trempe, G. (1975) in Human Tumor Cell Lines in Vitro (Fogh, J., ed), pp. 115-159, Plenum Press, New York
  29. Rodan, S. B., Imai, Y., Thiede, M. A., Wesolowski, G., Thompson, D., Bar-Shavit, Z., Shull, S., Mann, K., Roden, G. (1987) Cancer Res. 47, 4961-4966[Abstract]
  30. Forrest, S. M., Ng, K. W., Findlay, D. M., Michelangeli, V. P., Livesey, S. A., Partridge, N. C., Zajac, J. D., Martin, T. J. (1985) Calcif. Tiss. Int. 37, 51-56[Medline] [Order article via Infotrieve]
  31. Schnieke, A., Harbers, K., and Jaenisch, R. (1983) Nature 304, 315-320[Medline] [Order article via Infotrieve]
  32. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) J. Cell Biol. 96, 191-198[Abstract]
  33. Bateman, J. F., Mascara, T., Chan, D., and Cole, W. G. (1984) Biochem. J. 217, 103-115[Medline] [Order article via Infotrieve]
  34. Chan, D., Lamandé, S. R., Cole, W. G., Bateman, J. F. (1990) Biochem. J. 269, 175-181[Medline] [Order article via Infotrieve]
  35. Wake, S. A., and Mercer, J. F. B. (1985) Biochem. J. 228, 425-432[Medline] [Order article via Infotrieve]
  36. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  37. Tillet, E., Wiedemann, H., Golbik, R., Pan, T.-C., Zhang, R.-Z., Mann, K., Chu, M.-L., and Timpl, R. (1994) Eur. J. Biochem. 221, 177-185[Abstract]
  38. Specks, U., Mayer, U., Nischt, R., Spissinger, T., Mann, K., Timpl, R., Engel, J., and Chu, M.-L. (1992) EMBO J. 11, 4281-4290[Abstract]
  39. von der Mark, H., Aumailley, M., Wick, G., Fleischmajer, R., and Timpl, R. (1984) Eur. J. Biochem. 142, 493-502[Abstract]
  40. Wu, J. J., Eyre, D. R., and Slayter, H. S. (1987) Biochem. J. 248, 373-381[Medline] [Order article via Infotrieve]
  41. Bateman, J. F., Chan, D., Mascara, T., Rogers, J. G., Cole, W. G. (1986) Biochem. J. 240, 699-708[Medline] [Order article via Infotrieve]
  42. Lamandé, S. R., and Bateman, J. F. (1993) Matrix 13, 323-330[Medline] [Order article via Infotrieve]
  43. Dziadek, M., Darling, P., Bakker, M., Overall, M., Zhang, R.-Z., Pan, T.-C., Tillet, E., Timpl, R., and Chu, M.-L. (1996) Exp. Cell Res. 226, 302-315[CrossRef][Medline] [Order article via Infotrieve]
  44. Hagg, R., Hedbom, E., Mollers, U., Aszodi, S., Fassler, R., and Bruckner, P. (1997) J. Biol. Chem. 272, 20650-20654[Abstract/Free Full Text]
  45. Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O., Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649[Abstract/Free Full Text]
  46. Berg, R. A., Schwartz, M. L., and Crystal, R. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4746-4750[Abstract]
  47. Ripley, C. R., Fant, J., and Bienkowski, R. S. (1993) J. Biol. Chem. 268, 3677-3682[Abstract/Free Full Text]
  48. Kielty, C. M., Lees, M., Shuttleworth, C. A., Woolley, D. (1993) Biochem. Biophys. Res. Commun. 191, 1230-1236[CrossRef][Medline] [Order article via Infotrieve]
  49. Myint, E., Brown, D. J., Ljubimov, A. V., Kyaw, M., Kenney, M. C. (1996) Cornea 15, 490-496[Medline] [Order article via Infotrieve]
  50. Doege, K. J., and Fessler, J. H. (1986) J. Biol. Chem. 261, 8924-8935[Abstract/Free Full Text]
  51. Brass, A., Kadler, K. E., Thomas, J. T., Grant, M. E., Boot-Handford, R. P. (1992) FEBS Lett. 303, 126-128[CrossRef][Medline] [Order article via Infotrieve]
  52. Engel, J., Furthmayr, H., Odermatt, E., von der Mark, H., Aumailley, M., Fleischmajer, R., Timpl, R. (1985) Ann. N. Y. Acad. Sci. 460, 25-37[Abstract]
  53. Kuo, H. J., Keene, D. R., and Glanville, R. W. (1995) Eur. J. Biochem. 232, 364-372[Abstract]
  54. Zanussi, S., Doliana, R., Segat, D., Bonaldo, P., and Colombatti, A. (1992) J. Biol. Chem. 267, 24082-24089[Abstract/Free Full Text]
  55. Bonaldo, P., Russo, V., Bucciotti, F., Doliana, R., and Colombatti, A. (1990) Biochemistry 29, 1245-1254[Medline] [Order article via Infotrieve]


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