Identification, Purification, and Characterization of Cell-surface Retention Sequence-binding Proteins from Human SK-Hep Cells and Bovine Liver Plasma Membranes

doi:10.1074/jbc.270.4.1807

Identification, Purification, and Characterization of Cell-surface Retention Sequence-binding Proteins from Human SK-Hep Cells and Bovine Liver Plasma Membranes (*)

From the (1)Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104 and
(2)Monsanto Company, St. Louis, Missouri 63167

^¶ To whom correspondence should be addressed:
Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104
. Tel.: 314-577-8135; Fax: 314-772-1307.

Abstract

Cell-surface retention is a newly identified mechanism associated with the secretion of certain polypeptide growth factors and cytokines. This novel form of secretion appears to be mediated by cell-surface retention sequences (CRS) in the polypeptide molecules. To test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface retention, we identified and characterized the high-affinity binding sites on various cell types for I-labeled CRS peptide (sis) and CRS peptide (VEGF), each of which contained the putative CRS motifs of platelet-derived growth factor B (c-sis) and vascular endothelial cell growth factor, respectively. Scatchard plot analysis revealed a single class of high-affinity binding sites with K = 0.5-0.7 nM and 22,000-55,000 sites/cell. High-affinity binding activity could be demonstrated between pH 4.5 and 8.0, but was much greater below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were identified as 60-72-kDa proteins by ligand affinity labeling.

CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.

Footnotes

↵^§ Contributed equally to this work.
↵* This work was supported by National Institutes of Health Grant CA-38808 and by a grant from Monsanto Co. 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:

PDGF

platelet-derived growth factor

c-sis

cellular homolog of the viral oncogene v-sis

VEGF

vascular endothelial cell growth factor

CRS

cell-surface retention sequence(s)

CRS-BP

CRS-binding protein

HS-PG

heparan sulfate proteoglycan

EDAC

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl

BSA

bovine serum albumin

DMEM

Dulbecco's modified Eagle's medium

Mes

2-(N-morpholino)ethanesulfonic acid.
↵² C. Boensch, M.-D. Kuo, D. T. Connolly, S. S. Huang, and J. S. Huang, unpublished results.
- Received August 26, 1994.
- Revision received November 1, 1994.