Complete primary structure of the α1-chain of human basement membrane (type IV) collagen

A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natural sequence. However, in the position of the serine residue, some alterations were compatible with activity. Assay of peptides containing the structure Arg-Gly-Asp-X (where X = another amino acid residue) showed that an Arg-Gly-Asp-Val sequence predicted to be present in some, but not all, fibronectin molecules as a result of alternative RNA splicings could potentially create a second cell attachment site in those fibronectin polypeptide chains carrying that sequence. Other proteins with potentially active Arg-Gly-Asp-X sequences include several proteins that are known to interact with the cell surface. Among these are various types of collagens, thrombin, and discoidin, a slime-mold protein that may be involved in cell aggregation. The result presented here show that the arginine, glycine, and aspartic acid residues are absolutely required for the cell recognition, and that the surrounding amino acids may play a role in the expression of cell attachment activity in fibronectin and other proteins having this sequence. We suggest, based on these data, that this recognition mechanism may be common to a number of biological systems.

Monoclonal antibodies were produced against the recently described short chain cartilage collagen (type X collagen), and one (AC9) was extensively characterized and used for immunohistochemical localization studies on chick tissues. By competition enzyme-linked immunosorbent assay, antibody AC9 was observed to bind to an epitope within the helical domain of type X collagen and did not react with the other collagen types tested, including the minor cartilage collagens 1 alpha, 2 alpha, 3 alpha, and HMW-LMW. Indirect immunofluorescence analyses with this antibody were performed on unfixed cryostat sections from various skeletal and nonskeletal tissues. Only those of skeletal origin showed detectable reactivity. Within the cartilage portion of the 13-d- old embryonic tibiotarsus (a developing long bone) fluorescence was observed only in that region of the diaphysis containing hypertrophic chondrocytes. None was detectable in adjacent regions or in the epiphysis. Slight fluorescence was also present within the surrounding sleeve of periosteal bone. Consistent with these results, the antibody did not react with the cartilages of the trachea and sclera, which do not undergo hypertrophy during the stages examined. It did, however, lightly react with the parietal bones of the head, which form by intramembranous ossification. These results are consistent with our earlier biochemical analyses, which showed type X collagen to be a product of that subpopulation of chondrocytes that have undergone hypertrophy. In addition, either it or an immunologically cross- reactive molecule is also present in bone, and exhibits a diminished fluorescent intensity as compared with hypertrophic cartilage.