The recombinant transmembrane protein type XIII collagen is shown to reside on the plasma membrane of insect cells in a 'type II' orientation. Expressions of deletion constructs showed that sequences important for the association of three alpha1(XIII) chains reside in their N- rather than C-terminal portion. In particular, a deletion of residues 63-83 immediately adjacent to the transmembrane domain abolished the formation of disulfide-bonded trimers. The results imply that nucleation of the type XIII collagen triple helix occurs at the N-terminal region and that triple helix formation proceeds from the N- to the C-terminus, in opposite orientation to that of the fibrillar collagens. Interestingly, a sequence homologous to the deleted residues was found at the same plasma membrane-adjacent location in other collagenous transmembrane proteins, suggesting that it may be a conserved association domain. The type XIII collagen was secreted into insect cell medium in low amounts, but this secretion was markedly enhanced when the cytosolic portion was lacking. The cleavage occurred in the non-collagenous NC1 domain after four arginines and was inhibited by a furin protease inhibitor.

The complete primary structure of the mouse type XIII collagen chain was determined by cDNA cloning. Comparison of the mouse amino acid sequences with the previously determined human sequences revealed a high identity of 90%. Surprisingly, the mouse cDNAs extended further in the 5 direction than the previously identified human clones. The 5 sequences contained a new in-frame ATG codon for translation initiation which resulted in elongation of the N-terminal noncollagenous domain by 81 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the criteria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membrane, with a short cytosolic N-terminal portion and a long collagenous extracellular portion.These sequence data prompted us to generate antipeptide antibodies against type XIII collagen in order to study the protein and its subcellular location. Western blotting of human tumor HT-1080 cell extract revealed bands of over 180 kDa. These appeared to represent disulfide-bonded multimeric polypeptide forms that resolved upon reduction into 85-95-kDa bands that are likely to represent a mixture of splice forms of monomeric type XIII collagen chains. These chains were shown to contain the predicted N-terminal extension and thus also the putative transmembrane segment. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining were used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.The collagen family of proteins presently includes 19 types of collagen, and several additional proteins have collagen-like domains (1, 2). The collagens can be divided into two subgroups in terms of their structural and functional characteristics, the fibril-forming and the nonfibril-forming collagens. Members of the former group, i.e. types I-III, V, and XI, aggregate into prominent fibrillar structures in many collagen-containing tissues. These molecules are structurally homologous and characterized by a long, uninterrupted collagen triple helix. The other collagens are unable to form fibrils, and they show considerable diversity in structure, macromolecular organization, tissue distribution, and function. One common feature is that they all have one or more interruptions in the collagenous sequence. Several subfamilies can be distinguished among the nonfibril-forming collagens as follows: the network-forming collagens (types IV, VIII and X), fibril-associated collagens with interrupted triple helices (which include types IX, XII, XIV, XVI and XIX), a beaded filament-forming collagen (type VI), the family of types XV and XVIII collagens, and a collagen with a transmembrane domain (type XVII). The last mentioned collagen is distinct from the other family members, because it is not secreted into the extracellular matrix.Type XIII collagen is...

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine-and proline-rich (His/Pro-rich) domain.