A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.
We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.
Cooper
S
S
Department of Zoology, Oxford University, U.K.
Pera
M F
MF
Bennett
W
W
Finch
J T
JT
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D011508:Chondroitin Sulfate Proteoglycans / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000737:chemistry*; D006801:Humans; D007632:Keratan Sulfate / Q000737:chemistry / Q000302:isolation & purification*; D000071234:Lumican; D008854:Microscopy, Electron; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D011508:Chondroitin Sulfate Proteoglycans; C000604306:LUM protein, human; D000071234:Lumican; D007632:Keratan Sulfate