TYPE-XIII-COLLAGEN IS IDENTIFIED AS A PLASMA-MEMBRANE PROTEIN

The complete primary structure of the mouse type XIII collagen chain w as determined by cDNA cloning. Comparison of the mouse amino acid sequ ences with the previously determined human sequences revealed a high i dentity of 90%. Surprisingly, the mouse cDNAs extended further in the 5' direction than the previously identified human clones. The 5' seque nces contained a new in-frame ATG codon for translation initiation whi ch resulted in elongation of the N-terminal noncollagenous domain by 8 1 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the cri teria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membran e, with a short cytosolic N-terminal portion and a long collagenous ex tracellular portion. These sequence data prompted us to generate antip eptide antibodies against type XIII collagen in order to study the pro tein and its subcellular location. Western blotting of human tumor HT- 1080 cell extract revealed bands of over 180 kDa. These appeared to re present disulfide-bonded multimeric polypeptide forms that resolved up on reduction into 85-95-kDa bands that are likely to represent a mixtu re of splice forms of monomelic type XIII collagen chains. These chain s were shown to contain the predicted N-terminal extension and thus al so the putative transmembrane segment. Immunoprecipitation of biotinyl ated type XIII collagen from surface-labeled HT-1080 cells, subcellula r fractionation, and immunofluorescence staining were used to demonstr ate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.