A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen.
Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.
Koch
M
M
Department of Biophysical Chemistry, Biocenter of the University, Basel, Switzerland.
Bernasconi
C
C
Chiquet
M
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D003012:Microbial Collagenase; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011509:Proteoglycans / Q000378:metabolism* / Q000648:ultrastructure; D012867:Skin / Q000166:cytology; D014357:Trypsin
D010446:Peptide Fragments; D011509:Proteoglycans; D003094:Collagen; D004247:DNA; D014357:Trypsin; D003012:Microbial Collagenase