Multiple Forms of Chicken a3(VI) Collagen Chain Generated by Alternative Splicing in Type A Repeated Domains

Type VI collagen is a structurally unique component widely distributed in connective tissues. Its molecular structure consists of monomers that have the potential to assemble intracellularly into dimers and tetramers which, once secreted, can form microfilaments by end-to-end association. Individual monomers are composed of chains of Mr = ~140,000 (cd and a2) and >300,000 (or3). Type VI collagen molecules contain a short triple helix with large globular domains at both ends. These domains are made for their greatest part of repetitive units similar to type A repeats of von Willebrand Factor. The ot 3(VI) chain, contributing most of the mass of the NH~-terminal globule, appeared heterogenous both at the mRNA and protein level. Several ot3(VI)-specific clones that lack the sequences corresponding to repeats A8 and A6 were isolated from a chicken aorta cDNA library. Northern blot hybridization of poly (A+)-enriched RNA from chicken gizzard with cDNA fragments corresponding to several individual type A repeats showed that A8and A6-specific probes did not hybridize to the lower Mr transcripts. Clones spanning ~o20 kb of the 5'-end of the ot 3(VI) gene were isolated from a chicken genomic library and subjected to analysis by restriction mapping, Southern blotting, and selective sequencing of the intron-exon boundaries. At the most 5'-end of the gene an additional type A repeat (A9), previously undetected in eDNA clones, was identified. Furthermore, it was determined that the presumed signal peptide and repeats A9 through A6 are encoded within individual exons. Reverse transcription and polymerase chain reaction of aorta RNA suggested that a mechanism of alternative mRNA splicing by a phenomenon of exon skipping generates tx3(VI) isoform variants that contain different numbers of type A repeats. Immunohistochemistry of frozen sections of chicken embryo tissues with repeat-specific mAbs showed that an antibody directed against a conditional exon has a more restricted tissue distribution compared to an antibody against a constitutive exon. T YPE VI, one of the major collagens of connective tissues, is a component of 100-rim-long periodic microfilaments that are found at the surface of cells and around or between collagen fibers (von der Mark et al., 1984; Bruns, 1984; Bruns et al., 1986; Keene et al., 1988). The widespread occurrence of these thin fibrils in embryo (Bruns et al., 1986) and adult tissues (vonder Mark et al., 1984; Keene et al., 1988) and the diversity in localization, ranging from cartilage to soft tissues (Burgeson, 1988), are characteristic features of this collagen. The molecular mechanisms of microfilament formation are presently unknown but electron microscopic (Furthmayr et al., 1983) and biosynthetic studies (Engvail et al., 1986; Colombatti et ai., 1987; Colombatti and Bonaldo, 1987) have provided evidence that the polymerization process takes place intracellularly soon after synthesis and leads to the formation of disulfide-bonded dimers and tetramers. Furthermore, the individual chains do not seem to undergo proteolytic processing with removal of the large Nand Cpropeptides that do not represent precursor structures. The tetramers associate extracellularly by end-to-end to form the oligomeric microfilaments (Furthmayr et al., 1983). Recently, we (Bonaldo et al., 1989, 1990) and others (Koller et al., 1989; Chu et al., 1989) provided evidence that a major portion of the constituent chains of chicken and human type VI collagen consists of repeating units of ~ 200 residues that are closely related to the type A repeats of von Willebrand Factor (Shelton-Inloes et al., 1986). The most distinctive feature that emerged from the analysis of these sequences was the finding that ,,085 % of the or3 (VI) chain is represented by two types of similar repeating motifs, designated domains A and #/(Bonaido et ai., 1990). In a previous study Engvall et al. (1986) described the heterogeneity of the a3 (VI) chain present as three or more closely spaced bands in SDS-PAGE. The possibility was put forward that this heterogeneity was the consequence of posttranslational events. Similar discrete bands were detected by us after a short 7-min pulse (Colombatti et ai., 1987) and even after immunoprecipitation of tunicamycin and ot,ot'-dipyridyl-treated chicken embryo cells (Colombatti and Bonaldo, 1987). © The Rockefeller University Press. 0021-9525/90/11/2197/9 $2.00 The Journal of Cell Biology. Volume 111, November 199