Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lgtl 1 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the a2(VI) or al(V1) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encodes the a3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.Collagen type VI is a unique member within the family of collagenous proteins [l] and is characterized by a rather short triple helix (length 105 nm, M , = 120000), flanked on each side by a globular domain ( M , z 150000). These monomeric structures have the potential to form well-defined, disulfidelinked dimers and tetramers considered to be the building blocks of microfibrils which are abundant in the extracellular matrix [2]. Collagen VI was originally isolated from pepsin digests of tissues [3], a procedure which results in the loss of more than half of its original mass. Such materials were nevertheless valuable for chemical and electron microscope characterizations [4-71, which indicated that the chain fragments have M , = 40000 -70000. These studies eventually led to the proposal that collagen VI is composed of three constituent chains named al(VI), a2(VI) and a3(VI) [8]. Subsequent studies using antibodies raised against pepsin-solubilized collagen VI demonstrated that the genuine chain constituents are considerably larger, with M , = 110000-140000 19-151. A more recent investigation provided evidence that this size refers only to the al(V1) and a2(VI) chains, as a3(VI) is still larger (Mr = 200000) and is presumably synthesized in precursor form with M , = 250000 [16]. Previously biosynthetic studies demonstrated that a large peptide was closely associated with collagen VI, yet no data had shown that it contained collagenous sequences [17-201. In view of the various attempts to characterize the structure of collagen VI, only little is known about its biological roles [2] which may include the potential for cell-binding [21].A drawback in the studies of intact collagen VI has been the difficulty to isolate the material in amounts sufficient for In an effort to overcome some of these difficulties we have initiated studies on the identificati...
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