Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins.
Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)
Baeckström
D
D
Department of Medical Biochemistry, University of Göteborg, Sweden.
Hansson
G C
GC
Nilsson
O
O
Johansson
C
C
Gendler
S J
SJ
Lindholm
L
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis*; D018395:CA-19-9 Antigen; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003110:Colonic Neoplasms / Q000737:chemistry / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005732:Gangliosides / Q000032:analysis* / Q000276:immunology; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008970:Molecular Weight; D009077:Mucins / Q000737:chemistry* / Q000302:isolation & purification; D009368:Neoplasm Transplantation; D014183:Transplantation, Heterologous
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D018395:CA-19-9 Antigen; D002241:Carbohydrates; D000939:Epitopes; D005732:Gangliosides; D009077:Mucins; C064564:sialyl Le(a) ganglioside