Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis.
In the accompanying study (Cho, M., and Cummings, R. D. (1995) J. Biol. Chem. 270, 5198-5206), we reported that Chinese hamster ovary (CHO) cells synthesize galectin-1. We have now used several approaches to define the subcellular location and biosynthesis of galectin-1 in these cells. Galectin-1 was present on the cell surface, as assessed by immunofluorescent staining with monospecific antibody to the protein. Quantitation of the surface-localized galectin-1 was achieved by metabolically radiolabeling cells with [35S]Met/Cys and measuring the amount of lectin (i) sensitive to trypsin, (ii) accessible to biotinylating reagents, and (iii) accessible to the haptenic disaccharide lactose. By all three procedures, approximately 1/2 of the radiolabeled galectin-1 associated with cells was shown to be on the cell surface with the remainder intracellular. The kinetics of externalization of galectin-1 was monitored by pulse-chase radiolabeling, and it was shown that cells secrete the protein with a t1/2 approximately 20 h. The cell surface form of galectin-1 in CHO cells was active and bound to surface glycoconjugates, but lectin accumulating in the culture media was inactive. Lectin synthesized by mutant Lec8 CHO cells, which are unable to galactosylate glycoproteins was not found on the surface and quantitatively accumulated in the media in an inactive form. Taken together, our results demonstrate that galectin-1 is quantitatively externalized by CHO cells and can associate with surface glycoconjugates where the lectin activity is stabilized.
Cho
M
M
University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, Oklahoma City 73190.
Cummings
R D
RD
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003553:Cystine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism; D037483:Galectin 1; D006388:Hemagglutinins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D007150:Immunohistochemistry; D007700:Kinetics; D037102:Lectins / Q000096:biosynthesis*; D008715:Methionine / Q000378:metabolism; D008954:Models, Biological; D008970:Molecular Weight; D013347:Subcellular Fractions / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014357:Trypsin
D037483:Galectin 1; D006388:Hemagglutinins; D037102:Lectins; D013462:Sulfur Radioisotopes; D003553:Cystine; D008715:Methionine; D014357:Trypsin; D005690:Galactose