Galectin-1, a 
-Galactoside-binding Lectin in Chinese Hamster Ovary Cells
II. LOCALIZATION AND BIOSYNTHESIS (*)
- From the University of Oklahoma Health Sciences Center, Department of Biochemistry and Molecular Biology, Oklahoma Center for Molecular Medicine, Oklahoma City, Oklahoma 73190
Abstract
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 [
S]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, ≈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 t
≈ 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.
Footnotes
-
↵* This work was supported by National Institutes of Health Grant CA37626 (to R. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- CHO
-
Chinese hamster ovary
- PBS
-
phosphate-buffered saline (6.7 mM KH2PO4, 140 mM NaCl, pH 7.2)
- TPBS
-
phosphate-buffered saline containing 0.05% Tween 20
- SPB
-
PBS containing 14 mM 2-mercaptoethanol
- PAGE
-
polyacrylamide gel electrophoresis
- NHS
-
N-hydroxysulfosuccinimide
- BSA
-
bovine serum albumin.
-
- Received October 27, 1994.
- Revision received January 9, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
