©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Secretion and Matrix Assembly of Recombinant Type VI Collagen (*)

Alfonso Colombatti (1) (2)(§), Maria Teresa Mucignat (1), Paolo Bonaldo (3)

From the (1) Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, 33081 Aviano, the (2) Dipartimento di Scienze e Tecnologie Biomediche, Universit di Udine, 35100 Udine, and the (3) Istituto di Istologia ed Embriologia, Universit di Padova, 35100 Padova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A monomer of type VI collagen is composed of three different chains of 140 (1), 130 (2), and 250-350 kDa (3). Monomers assemble into dimers (6 chains) and tetramers (12 chains) that are stabilized by disulfide bonds and, once associated one to another, give rise to a microfilamentous network in close apposition with cell surfaces and banded collagen fibers. We have derived murine NIH/3T3 cell lines that were transfected with the cDNAs for the three chains and that constitutively expressed chicken type VI collagen. Cotransfection was efficient because, in three out of six isolated cell lines, all chicken chains were expressed. Southern blotting demonstrated that several copies of each cDNA were integrated approximately in equal number. Expression of the three polypeptide chains was consistent with the levels of the respective mRNAs. The three chicken chains assembled by disulfide bonding to form correctly folded triple helical aggregated composites with sizes corresponding to type VI collagen monomers, dimers, and tetramers. These functional recombinant assemblies were secreted and became incorporated into the extracellular matrix, where they formed an extensive fibrillar network.


INTRODUCTION

Type VI collagen is a component of a microfilamentous meshwork (1-4) that is believed to play a significant role in cell-matrix interactions. Unique among the collagens discovered to date, type VI collagen constituent chains have significantly different sizes. The 1(VI) and 2(VI) chains are about 130-140 kDa; the 3(VI) chain is much larger, ranging from 250 to 350 kDa (5, 6, 7, 8) . Cloning and sequencing of the cDNAs for the chicken (9, 10, 11) and human (12, 13) chains have shown that 1(VI) and 2(VI) chains consist of a short collagenous sequence flanked at the N and C termini by one and two type A modules, respectively. A major portion of the additional sequences of the 3(VI) chain consists of eight (11, 13) or nine (14, 15) type A modules. Furthermore, the 3(VI) chain differs from the other two chains due to the presence of unique additional sequences at the C terminus (13, 16) .

Tissue extraction and biosynthetic studies have demonstrated that the three chains occur in stoichiometric proportions in a 1:1:1 ratio (17-19). In vitro studies have shown that type VI collagen has a peculiar pathway of intracellular assembly; association of the three chains in a type VI collagen monomer is followed by the formation of S-S bonded dimers (6 chains) and tetramers (12 chains) before secretion (19, 20) .

In an effort to understand the biology of type VI collagen assembly and cell-matrix interactions, a model system was initially developed in which murine NIH/3T3 cells were stably transfected with cDNAs encoding chicken type VI collagen chains. Cell lines were obtained that constitutively express the individual chicken chains; no self-association was observed with 1(VI) nor with 2(VI) chains, which were secreted as single polypeptides. Instead, chimeric chicken/murine type VI collagen molecules were detected in cell lines transfected with the chicken 3(VI) cDNA (21) .

In the present study, NIH/3T3 cells were successfully cotransfected with the cDNAs (3.15, 3.2, and 8.1 kb)() coding for the three chicken type VI collagen chains and for a selectable marker. Stable coexpression of all three chains was obtained, and the three transfected chains were assembled into pepsin-resistant type VI collagen. These molecules were secreted as monomers and higher order multimers of the proper size and became deposited into the extracellular matrix (ECM). This approach demonstrates for the first time the production of a recombinant nonfibrillar collagen composed of three genetically different chains.


MATERIALS AND METHODS

Antibodies

The following rabbit polyclonal antisera were used: AS-5, specific for chicken type VI collagen (19) ; AS-72, specific for murine type VI collagen; and AS-46 and AS-47, specific for the chicken 1(VI) and 2(VI) chains, respectively.() Tissue culture supernatants of the mAbs 111A3, 116A8, 192C2, and 76G11, specific for the chicken 3(VI) chain, and mAb 108F3, specific for the chicken 1(VI) chain (22),() were used; ascites of the antifibronectin mAb HB91 (ATCC) was also used.

Expression Vectors

Construction of the 1(VI) full-length cDNA (3.15 kb) and insertion into the SV40 early replacement vector pSV23 plus (23) was described previously (24) . The 2(VI) full-length cDNA (3.2 kb) was constructed from four partial cDNA clones (7a, 7u, 4a0, and 2a0) obtained from Dr. B. Trüeb (Zurich, Switzerland). The clones were digested with XbaI and AccI (7a), AccI and EcoRI (7u), EcoRI (4a0), or EcoRI and BglII (2a0) and were ligated in the M13mp19 vector. An XbaI-NheI full fragment was then excised from M13 and inserted into the pSV23 plasmid. The construction of 3(VI) cDNA (8.1 kb) required the use of four cDNA clones that were ligated two by two (pB118 with pB4 and pB32 with pB72) in the M13 mp 18 vector. The two constructs were then excised from M13 and ligated into pSV23 digested with SmaI. The pB118 cDNA clone used for this construction lacked the modules A8 and A6, which are coded by individual exons that can undergo alternative splicing (14). A schematic drawing of these steps is shown in Fig. 1.


Figure 1: Schematic diagram of the pSV-based vectors. The construction of chicken 1(VI), 2(VI), and 3(VI) cDNA is shown. The cDNA clones used for the construction were described previously (9, 10, 14, 24). Polylinker restriction sites are within parentheses. A, AccI; B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII, K, KpnI; N, NheI; P, PstI; S, SalI; Sm, SmaI; X, XbaI. P, t, and pA indicate the SV40 early promoter, the small-t intron, and the polyadenylation site, respectively.



Cell Culture, DNA Transfection, and Cell Line Selection and Identification

NIH/3T3 cells were grown in Dulbecco's modified minimum essential medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamycin. DNA transfections were performed using the calcium phosphate coprecipitation procedure of Graham and van der Eb (25) as modified by Chen and Okayama (26) , and DNA was purified by double sedimentation in a cesium chloride gradient. In a typical experiment, 6 10 cells plated 24 h earlier in a 60-mm Petri dish were cotransfected for 20 h at 37 °C with 1.5 µg each of supercoiled DNA of pSV23-1(VI) and -2(VI) and 3 µg of supercoiled DNA of pSV23-3(VI) plus 40 ng of a plasmid containing the neo gene (pWLneo, Stratagene, La Jolla, CA). The cells were maintained overnight in Dulbecco's modified minimum essential medium supplemented with 10% fetal calf serum in a 3% CO atmosphere, and the following day the medium was changed and the CO concentration was raised to 5%. Selection was carried out in 500 ng/ml of G418 (Sigma), and individual colonies were isolated and grown into separate cell lines.

Northern and Southern Blotting

High molecular weight DNA was isolated from transfected cell lines following established procedures (27) , and 3 µg were digested to completion with restriction enzymes. The fragments were resolved on a 0.7% (w/v) agarose gel, transferred onto Hybond nylon membrane (Amersham International, Buckinghamshire, UK), and hybridized to cDNA probes of the 1(VI), 2(VI), and 3(VI) chains labeled with [-P]dCTP (Amersham) by the random primed oligolabeling method (28) .

Total RNA was isolated by guanidine isothiocyanate extraction (29) . Electrophoresis of the RNA was performed on a 0.7% (w/v) agarose gel containing 2.3 M formaldehyde in MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA) for 8 h at 150 V using 20-cm-long plates. RNA was then transferred onto nitrocellulose filters and hybridized with [-P]dCTP-labeled cDNA probes specific for the different type VI collagen chains.

The filters were hybridized at 68 °C overnight in 5 NaCl/phosphate/EDTA (180 mM NaCl, 10 mM phosphate, 1 mM EDTA, pH 7.7) (buffer A) containing 5 Denhardt's solution, 0.5% (w/v) SDS, and 100 µg/ml salmon sperm DNA. After washing in 2 NaCl/phosphate/EDTA for 5 min at room temperature, in 1 buffer A plus 1% SDS for 30 min at 65 °C, and in 0.1 NaCl/phosphate/EDTA for 30 min at room temperature, the filters were exposed to -max Hyperfilms (Amersham).

Metabolic Labeling and Immunoprecipitation

Cells were metabolically labeled in methionine-free Dulbecco's modified minimum essential medium containing 1% dialyzed fetal calf serum. Labeling with [S]methionine (800 Ci/mmol, Amersham) at 100-300 µCi/ml was carried out in the presence or the absence of 50 µg/ml ascorbate. After 3-4 h the cell layer was solubilized for 30 min at 0 °C in extraction buffer with the following final concentrations: 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 25 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 mMN-ethylmaleimide, and 1 mMpara-aminobenzamidine. When needed, the medium was brought to extraction buffer. The medium and the cell lysate were clarified by centrifugation at 10,000 g and used for immunoprecipitation. The samples were first precleared by incubation with 2 µl of antifibronectin mAb (HB91) followed by incubation with 100 µl protein A-Sepharose (Pharmacia Biotech Inc., Uppsala, Sweden) for 1 h at 0 °C. After centrifugation for 15 min at 3,000 g, 2 µl of AS-5 or 100 µl of mAbs against the chicken 3(VI) chain and 50 µl of protein A-Sepharose were added to the supernatant, and the incubation proceeded for 4-6 h. The samples were centrifuged, and the precipitates were washed extensively in extraction buffer prior to dissolving in Laemmli sample buffer in the presence or in the absence of 5% (v/v) 2--mercaptoethanol (30) .

In some instances, before proceeding with the immunoprecipitation with anti chicken type VI collagen antibodies, the medium and the cell lysate were preincubated with AS-72. Immunoprecipitated material was resolved by SDS-PAGE on 6% (v/v) or 3-10% linear gradient polyacrylamide slab gels using the buffer system of Laemmli and was exposed to MP hyperfilms (Amersham).

Pepsin Digestion andI Labeling

Cultures of D23/3 cells in Dulbecco's modified minimum essential medium containing fetal calf serum and supplemented for 2-3 days with ascorbate (50 µg/ml) were washed with serum-free medium and then maintained overnight in serum-free medium. The culture medium was collected, dialyzed against PBS, and labeled with I by the chloramine-T method. Labeled proteins were separated from free iodine by gel filtration and brought up into 0.5 M acetic acid for treatment with 100 µg/ml pepsin for 3 h at 4 °C. The sample was dialyzed extensively against PBS, and aliquots were immunoprecipitated with AS-5, AS-46, and AS-72. Immunoprecipitated material was resolved by SDS-PAGE on 8% (v/v) polyacrylamide slab gels and exposed to MP Hyperfilms.

Matrix Deposition

Matrix deposition of transfected chicken type VI collagen polypeptides was analyzed by indirect immunofluorescence. NIH/3T3 cell clones expressing the three chicken type VI collagen chains were cultured on chamber slides (A/S Nunc, Roskilde, Denmark) until almost confluent, washed with PBS, and fixed for 30 min at room temperature with iced 4% paraformaldehyde in PBS. Slides were incubated for 60 min at 4 °C with primary antibodies diluted in PBS containing 1% BSA in a moist chamber. Subsequent incubations were with rabbit anti-mouse or goat anti-rabbit fluorescein- or rhodamine-conjugated antibodies (A/S Dako, Glastrup, Denmark). Stained slides were mounted in glycerol and examined with an epifluorescent Leitz microscope using a 25 phase/fluorescence objective. Slides were photographed with Ilford HP5 400 (Ilford, Ltd., Mobberley Cheshire, UK).


RESULTS

Establishment of NIH/3T3 Cell Lines That Coexpress the Three Chains of Chicken Type VI Collagen

NIH/3T3 cells were cotransfected with the cDNAs coding for chicken type VI collagen chains along with the neo gene. Cells were put into selective G418 medium, and, after 14 days, 10 colonies were isolated and 6 grew into individual stable cell lines. The expression of chicken type VI collagen chains was investigated by immunostaining permeabilized cells with antibodies specific for the individual chicken type VI collagen chains. This screening (data not shown) suggested that three cell lines (D10/1, D23/3, and D24/1) coexpressed all transfected chains, two cell lines (D23/2 and D28/1) coexpressed only the 2(VI) and 3(VI) chains, and one cell line (D26/1) coexpressed the 1(VI) and 3(VI) chains (data not shown). Next, the six cell lines were metabolically labeled with [S]methionine, and the cell lysates were immunoprecipitated with AS-5 (Fig. 2). No material was immunoprecipitated from normal NIH/3T3 cells, confirming that the antibody was specific for chicken type VI collagen and did not recognize murine type VI collagen. The immunoprecipitates from the six transfected cell lines contained several bands of different intensity and migration. Five cell lines showed bands, prominent in D10/1, D23/2, and D26/1, at about 240 kDa, consistent with the size expected for the transfected chicken 3(VI) cDNA. In the D28/1 cell line, the 240-kDa band was absent, whereas a band migrating at about 220 kDa was immunoprecipitated. A band of similar size also was detected in D24/1 cells. Lower bands migrating in the range of 130-140 kDa as expected for 1(VI) and/or 2(VI) chains were present in all six cell lines.


Figure 2: Identification of polypeptides immunoprecipitated from cell lines cotransfected with chicken 1(VI), 2(VI), and 3(VI) cDNAs. Cells were metabolically labeled with 100 µCi/ml [S]methionine for 3 h. Cells were collected, and cell lysates were immunoprecipitated with AS-5 polyclonal antiserum to chicken type VI collagen and resolved in a 6% polyacrylamide gel under reducing conditions. On the right is the migration of the transfected chicken type VI collagen chains, and on the left the migration of molecular weight markers is indicated. In this experiment the preabsorption step with antifibronectin antibody was not exhaustive, and, consequently, some fibronectin contaminated the immunoprecipitates.



Two cell lines (D23/3 and D24/1) coexpressing all three transfected cDNAs were successfully subcloned, and their DNA and RNA were isolated. To demonstrate the presence of each of the integrated cDNAs, high molecular weight DNA was digested and analyzed by Southern blotting. Under the stringent conditions used, no hybridization with endogenous murine type VI collagen genes was detected. On the other hand, both cell lines incorporated several copies of all three chicken cDNAs (Fig. 3).


Figure 3: Southern blot analysis of D23/3- and D24/1-transfected cell lines. Each lane was loaded with 10 µg of high molecular weight DNA digested with BclI. Individual strips were hybridized to [-P]dCTP-labeled cDNA of full-length chicken 1(VI) cDNA, 2(VI) cDNA, and clone pB10(3) (11). On the left the migration of DNA markers is indicated.



Because the sizes of the bands were ambiguous in the first series of immunoprecipitation, Northern blot analysis was carried out to control for the sizes of the transcripts (Fig. 4). Both subcloned D23/3 and D24/1 cell lines showed 1(VI) and 2(VI) transcripts of the proper size (about 3.2 kb). The D23/3 cell line had a properly sized 3(VI) chain transcript of about 8.0 kb, whereas in the D24/1 cell line the 3(VI) transcript was of slightly smaller size (about 7.0 kb). In addition, hybridization with the 3(VI)-specific probe revealed in both cell lines a prominent band at about 5.4 kb and, in D23/3 cells, also a minor signal at about 3.0 kb. It is possible that these smaller RNAs are transcribed from incorrectly integrated cDNAs or that they are the result of aberrant splicing.


Figure 4: Northern blot analysis of D23/3- and D24/1-transfected cell lines. Each lane was loaded with 5 µg of total RNA. Individual strips were hybridized to [-P]dCTP-labeled full-length chicken 1(VI) cDNA, 2(VI) cDNA, and clone pB10(3) (11). On the left the migration of RNA markers is indicated.



Cell Lines Constitutively Expressing ``Variant'' Chicken 3(VI) Chains

Immunoprecipitation and Northern blot analyses revealed variant 3(VI) mRNAs and polypeptides in some cell lines. To further investigate these aberrantly sized chicken 3(VI) chains, the D28/1, D23/3, and D24/1 cell lines were metabolically labeled, and the cell lysates were immunoprecipitated with mAbs specific for different domains of the chicken 3(VI) chain (Fig. 5). In the D28/1 cell line, AS-5 immunoprecipitated two major bands, one at about 220 kDa and one at 130 kDa (Fig. 5, lane 2, and also see Fig. 2, lane 7). Accordingly, the D28/1 cells expressed a correct 2(VI) mRNA (about 3.2 kb) and an aberrant 3(VI) mRNA of about 7.0 kb (not shown). In addition, the chicken 3(VI)-specific mAbs 192C2 and 116A8 immunoprecipitated the 220 kDa band nearly exclusively (Fig. 5, lanes 5 and 7), whereas neither mAb 111A3 nor 76G11 immunoprecipitated any polypeptide from D28/1 cells, suggesting that their epitopes are not present or not exposed in this smaller 3 (VI) polypeptide. Instead, a polypeptide of about 220 kDa was recognized by mAb 111A3 in D24/1 cells (Fig. 5, lane 9, and also see Fig. 2, lane 6), suggesting that this 3(VI) form differs from the similarly sized 220-kDa 3(VI) polypeptide of D28/1 cells. mAb 111A3 immunoprecipitated from D23/3 cells a polypeptide at about 240 kDa, which is the size expected for the correct 3(VI) transfected cDNA (Fig. 5, lane 8, and also see Fig. 2, lane 4). In this experiment a longer 6% gel was run, and the chicken and murine 1(VI) and 2(VI) chains could be resolved. In this gel it was evident that both chicken and murine bands were coimmunoprecipitated in D23/3 and D24/1 cells. In addition, mAb 111A3 was able to immunoprecipitate from both D23/3 and D24/1 cell lines a heavily labeled polypeptide of about 160 kDa that migrated slightly above the 1(VI) and 2(VI) chains (Fig. 5, lanes 8 and 9). A similarly sized polypeptide was immunoprecipitated by mAbs 111A3 and 76G11 also from a cell line (C11B7) transfected only with the 3(VI) cDNA (Fig. 5, lanes 10 and 12) (21) . This set of immunoprecipitations indicates that there is a certain degree of heterogeneity in the nature of the 3(VI) chains produced by different cell lines.


Figure 5: Polypeptides immunoprecipitated from transfected cell lines by chicken 3(VI) chain-specific monoclonal antibodies. D28/1, D23/3, D24/1, and C11B7 (24) cells were metabolically labeled for 3 h with 100 µCi of [S]methionine, and cell lysates were immunoprecipitated with mAbs to chicken 3(VI) collagen chain (111A3, 192C2, 76G11, and 116A8), with preimmune serum (NRS, lane 3), or with polyclonal antiserum against chicken type VI collagen (AS-5, lane 2) and resolved in a 6% (w/v) polyacrylamide gel under reducing conditions. The arrowheads indicate the migration of the transfected chicken type VI collagen chains, and the dots indicate the migration of the 160-kDa truncated 3(VI) form. On the left the migration of molecular weight markers is shown. Anti-FN, mAb HB91 against fibronectin.



Assembly, Secretion, and Matrix Deposition of Transfected Chicken Type VI Collagen

Type VI collagen chains usually form stable (1(VI), 2(VI), and 3(VI)) monomers that assemble intracellularly to give rise to S-S bonded dimers (6 chains) and tetramers (12 chains). To study the assembly of the transfected chicken chains, we first assessed that extensive and repeated immunoprecipitations depleted all of the murine chains from D23/3 cell lysates, leaving behind polypeptides that could be specifically immunoprecipitated with AS-5 (Fig. 6A, lane 2) but not with AS-72 (Fig. 6A, lane 3). D23/3 cells were selected for these experiments because they synthesize type VI chains of the correct size. Next, in a similar type of experiment we evaluated the extent of assembly of chicken type VI collagen chains in cell lysates. Aliquots of the immunoprecipitate were run in SDS-PAGE in the absence of 2--mercaptoethanol, and, under these conditions, most of the material consisted of larger forms that barely entered the 6% separating gel (Fig. 6B, lane 5).


Figure 6: Assembly and secretion of transfected type VI collagen. A, preclearing with AS-72. Cell lysates of metabolically labeled D23/3 were subjected to five cycles of immunoprecipitations with AS-72. After these extensive preclearing steps, the supernatant was aliquoted and immunoprecipitated with preimmune serum (NRS, lane 1), AS-5 (lane 2), or AS-72 (lane 3). B, assembly. Aliquots of the cell lysate immunoprecipitated with AS-5 as in lane 2 and were resolved under reducing (lane 4) or nonreducing (lane 5) conditions. A dotted line indicates the top of the separating gel. The migration of the transfected chicken chains and of molecular weight markers is shown. C, secretion. Cell medium of D23/3 cells metabolically labeled for 4 h with 300 µCi of [S]methionine in the presence of 50 µg/ml ascorbic acid was precleared with one immunoprecipitation cycle of AS-72. The supernatant was immunoprecipitated with AS-5 (lanes 6 and 9), followed by AS-72 (lane 7). An aliquot of cell medium was immunoprecipitated with HB91 antifibronectin mAb (lane 8), and the migration of fibronectin served as a marker in the nonreduced gel. The immunoprecipitates were analyzed in a 3-10% polyacrylamide linear gradient without stacking gel under reducing or nonreducing conditions. A dotted line indicates the top of the gel. FN-M and FN-D indicate the migration of monomers and dimers of fibronectin, respectively. VI-M, VI-D, and VI-T indicate the migration of type VI collagen monomers, dimers, and tetramers, respectively. 1(VI)c, 2(V)c, 3(VI)c, 1(VI)m, 2(VI)m, and 3(VI)m indicate the migration of the chicken (c) and murine (m) chains of type VI collagen.



Secretion of chicken type VI collagen assemblies was studied in D23/3 cells labeled in the presence of ascorbate. The cell medium was immunoprecipitated with AS-5, and the immunoprecipitate was run under reducing and nonreducing conditions in a 3-10% linear gradient SDS-PAGE without stacking gel in order to resolve also the larger assembled composites (Fig. 6C). In the nonreduced gel, the major polypeptide of 240 kDa corresponding to the chicken 3(VI) chain and other bands in the range of 130-140 kDa were strongly decreased. Instead, a larger form (about 500 kDa) slightly above the fibronectin dimer and more heavily labeled higher bands were detected. The sizes of these disulfide-bonded aggregates are similar to the sizes of properly assembled type VI monomers (about 500 kDa), dimers (about 1,000 kDa), and tetramers (about 2,000 kDa). To avoid the extensive immunodepletion of the murine type VI collagen that could eliminate coassociated chicken type VI collagen forming the higher order structures present in the cell medium, the preclearing steps with AS-72 were not very extensive. As a consequence, the pattern of chains immunoprecipitated from the cell medium was more complex than the pattern of chains immunoprecipitated from the cell lysate (see Fig. 6B), especially in the range of 130-140 kDa. In this case, when the supernatant from the first immunoprecipitation with AS-5 was subjected to a second immunoprecipitation with AS-72, some murine type VI collagen could still be revealed (Fig. 6C, lane 7).

The demonstration that transfected chicken type VI polypeptides apparently assembled to form S-S bonded tetramers suggests but does not prove that these composites are stable and have a proper triple helical conformation in their collagenous domain. The low methionine content of the triple helix and the amounts of transfected chicken type VI collagen secreted by [S]methionine-labeled D23/3 cells prevented the elucidation of this matter, because no polypeptides corresponding to the pepsin form of type VI collagen could be detected. Therefore, serum-free medium from ascorbate-treated D23/3 cells was collected, labeled with I, brought to 0.5 M acetic acid, and digested with pepsin. This analysis was facilitated by the notion that chicken and murine 1(VI) pepsinized chains migrate at distinct positions in SDS-PAGE (18) . As shown in Fig. 7, not only AS-5 (lane 2), which recognizes all three chicken chains, but also AS-46 (lane 4), which is specific for only the chicken 1(VI) chain, immunoprecipitated three pepsin-resistant fragments. With both antisera, the mobility of the 1(VI) band and also of the 3 (VI) band was slower compared with the mobility of the bands detected when the sample was immunoprecipitated with AS-72 (Fig. 7, lane 3). Although a low contamination of murine chains also was present in lanes 2 and 4 (Fig. 7), these results demonstrate that recombinant chicken type VI collagen can form stable triple helices.


Figure 7: Polypeptides immunoprecipitated from pepsin-digested D23/3-secreted material. Type VI collagen was immunoprecipitated with normal rabbit serum (NRS, lane 1), AS-5 (lane 2), AS-72 (lane 3), or AS-46 (lane 4). On the left the migration of molecular weight markers is indicated.



The ability of D23/3 cells to incorporate the secreted recombinant chicken type VI collagen into their ECM was investigated by staining D23/3 cells with different combinations of polyclonal antisera and mAbs that react with chicken or murine type VI collagen and with fibronectin. The results are shown in Fig. 8. Cells incubated with preimmune serum or an unrelated mAb showed only background fluorescence (not shown). Also negative were untransfected NIH/3T3 cells incubated with AS-5 (Fig. 8b). In contrast, strong immunofluorescence in a fibrillar pattern was detected when D23/3 cells were incubated with the AS-5 or with antibodies specific for each of the transfected chicken chains (Fig. 8, c, d, e, and g). This staining pattern was superimposable with the pattern of endogenous murine type VI collagen (Fig. 8, f and h). The incorporation of heterologous chicken type VI collagen into the ECM of NIH/3T3 cells indicates that transfected chicken type VI collagen assemblies are competent for matrix formation.


Figure 8: Immunofluorescence staining of D23/3 cells. Cells were cultured in medium containing ascorbic acid for 4 days and then fixed and stained with single antibodies or combinations (e and f or g and h) of various antibodies. NIH/3T3 cells were stained with AS-5 polyclonal antiserum specific for the chicken type VI collagen (b). D23 cells were stained with AS-72 anti murine type VI collagen (a and h), mAb 111A3 anti chicken 3(VI) chain (c and g), or AS-47 anti chicken 2(VI) chains (d). Double immunofluorescence was with mAb 108F3 anti chicken 1(VI) chain (e) or AS-72 (f) and with mAb 111A3 (g) or AS-72 (h). Fluorescein-conjugated anti-mouse Ig and rhodamine-conjugated anti-rabbit Ig were used as secondary reagents.




DISCUSSION

In this study, we have constructed cDNAs encoding the chicken 1(VI), 2(VI), and 3(VI) collagen chains and show that these three exogenous polypeptides can be constitutively expressed in murine NIH/3T3 cells in the forms of properly folded pepsin-resistant type VI collagen monomers and higher order disulfide-bonded multimers. Cotransfection with a high ratio of the chicken type VI cDNAs versus the neo selectable marker gene (about 30:1) resulted in the efficient and stable introduction of all cDNAs; three out of six selected cell lines expressed all three chains of chicken type VI collagen. Furthermore, in at least one cell line, heterologous recombinant chicken type VI collagen was secreted and incorporated into the ECM. The immunofluorescence pattern obtained with antibodies specific for the chicken and murine type VI collagen was superimposable and suggests that molecules made only of chicken or murine chains or chimeric chicken/murine type VI collagen might coassemble and be deposited.

Production of chicken type VI collagen assemblies was confirmed by preclearing experiments in which the murine type VI collagen present in cell lysates was specifically and completely removed, allowing the recovery of pure transfected heterologous chicken type VI collagen. The preclearing steps could not be as extensive with cell medium, because in this case also the chicken molecules were removed. Secreted type VI collagen multimers are larger than those present in cell lysates (21), and it is likely that chicken molecules coassemble with secreted murine molecules in larger assemblies and are more easily removed by the preclearing steps. To analyze secreted molecules it was necessary to compromise between detecting a discrete but only partially purified chicken type VI collagen signal and no signal at all. However, when a more sensitive I labeling of pepsin-resistant secreted material was used, polypeptides largely corresponding to chicken type VI collagen were specifically immunoprecipitated. The resistance to pepsin digestion demonstrated the triple helical conformation of recombinant chicken type VI molecules. Antibodies specific for the 1(VI) chicken chain were able to immunoprecipitate composites including mainly chicken 2(VI) and 3(VI) chains, indicating minimal interference from endogenous murine type VI polypeptides in the formation of chicken type VI monomers. Because the acetic acid treatment necessary for pepsin digestion disrupts all of the noncovalent bonds, nearly pure chicken macromolecular assemblies were immunoprecipitated. On the other hand, chimeric assemblies were more easily detected in immunoprecipitates made with molecules kept under native conditions. Therefore, the contribution of some chimeric murine/chicken molecules (i.e. murine 1(VI) and 2(VI) and chicken 3(VI)) also should be considered in interpreting the immunodetection of collagen type VI in the deposited ECM. However, the presence of chimeric assemblies will not undermine the conclusion that at least a fraction of deposited type VI collagen is solely of chicken origin.

The synthesis of endogenous type VI collagen in NIH/3T3 cells renders more complex the analysis of transfected cell lines. In preliminary experiments we have attempted to stably transfect with chicken type VI collagen cDNAs cell lines that do not produce endogenous type VI collagen, but we have so far failed to demonstrate assembly and secretion of transfected chains. It is possible that cells that already synthesize and secrete their own type VI collagen also favored the assembly and deposition into the ECM of the transfected chicken chains. Previously reported cell systems for analysis of transfection and full expression of type I collagen DNAs made use of cell lines that retained (31, 32) or did not retain (33) endogenous expression of one of the two chains of type I collagen. Secretion of trimers of type XII collagen was achieved in HeLa cells (34) , which are known to synthesize only low levels of type IV and type V collagens (35). In these latter studies, only the formation of a proper triple helix was reported, whereas the ECM deposition of heterologous recombinant collagens was not investigated.

One observation we have made frequently is that, in addition to the correct 8.0-kb 3(VI) mRNA signal and the 240-kDa 3(VI) polypeptide band, shorter messages in the range of 5.4-7 kb and polypeptides in the range of 160-220 kDa, which are present together with or instead of the properly sized molecules, are detected. The 5.4-kb mRNA band has been detected in most lines, and, because of its frequency, it seems unlikely that this shorter mRNA is transcribed from incorrectly integrated cDNAs. Alternatively, the 5.4-kb transcripts and the 160-kDa polypeptides might be due to aberrant splicing from cryptic splice donor sites of the 3(VI) cDNA, as has been reported in other experimental systems (36, 37, 38, 39) . The inability of some antibodies to immunoprecipitate the 160-kDa truncated 3(VI) polypeptide could depend on the localization of the recognized epitopes that are localized in the triple helix (AS-5) or in the C terminus (mAbs 116A8 and 192C2). This suggests that the truncated 160-kDa polypeptide is comprised of the N-terminal globular end of the 3(VI) chain. The 160-kDa polypeptide does not associate with 1(VI) and 2(VI) chains, and, therefore, it is likely that this smaller 3(VI) form lacks some sequences/domains that are involved in the association with the other two chains. It is noteworthy that a recombinant fragment of the human 3(VI) chain that includes all of the N-terminal globule and that corresponds to a large extent to the present 160 kDa polypeptide displayed several functional interactions, including binding sites for heparin, for hyaluronan, and also for the triple helical domain of type VI collagen (40) .

The present model offers a tool for detailed mutational analysis and the production of recombinant type VI collagen molecules of predefined composition to clarify the contribution of their globular non-triple helical domains in cell adhesion and migration phenomena (41, 42) . Currently, we are trying to establish stable cell lines with more efficient expression of transfected type VI collagen molecules in order to enable a more thorough structure-function analysis in the complex process of type VI collagen assembly and deposition.


FOOTNOTES

*
These studies were supported by the Consiglio Nazionale delle Ricerche ``Progetto Applicazioni Cliniche della Ricerca Oncologica'' and the Associazione Italiana per le Ricerche sul Cancro. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, Via Pedemontana Occidentale 12, 33081 Aviano, Italy. Tel.: 39-434-659301; Fax: 39-434-659428.

The abbreviations used are: kb, kilobase(s); ECM, extracellular matrix; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; mAb, monoclonal antibody.

A. Colombatti, M. T. Mucignat, and P. Bonaldo, unpublished data.

A. Colombatti, R. Doliana, M. T. Mucignat, D. Segat, S. Zanussi, C. Fabbro, and L. Magris, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Francesco Bucciotti for excellent technical assistance in the preparation of the vectors, Dr. Roberto Perris for reading the manuscript, and Antonella Moro for typing the manuscript. We thank also Dr. Beat Trüeb, Eidgenössische Technische Hochschule, Zurich, Switzerland for supplying us with clones of chicken 2(VI) and Dr. Danny Huylebroeck for providing the pSV23 vector.


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