Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6843-6847, September 1990 Cell Biology A major endothelial plasmalemmal sialoglycoprotein, gp6O, is immunologically related to glycophorin (receptors/capillary permeability/vasculitis/kems/serum albumin) JAN E. SCHNITZER*, JEFFREY B. ULMERt, AND GEORGE E. PALADE* Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Contributed by George E. Palade, June 15, 1990 ABSTRACT Glycophorins, the major sialoglycoproteins the study of transmembrane sialoglycoproteins. They have of red blood cells in many species, are generally considered to been isolated from various species and shown to contain up be specific to erythroid cells. Using polyclonal antibodies to 70%o (wt/wt) carbohydrate, present primarily as 0-linked directed against mouse glycophorin (agp), we have identified oligosaccharides. The function of glycophorins has not been a glycoprotein antigenically related to glycophorin on the established. Since they are heavily sialylated and usually surface of bovine and rat cultured endothelial cells. Immuno- contain up to 70%o of the sialic acid of the cell, one of their blotting with agp identified a single 60-kDa polypeptide on functions could be to restrict low-affinity, nonspecific inter- transfers of SDS/polyacrylamide gels of solubilized confluent actions at the cell surface. Indeed, sialic acid residues on the endothelial monolayers. In addition, a 60-kDa polypeptide was cell surface appear to prevent nonspecific hemagglutination, immunoprecipitated by agp from lysates of 12I-labeled intact erythrocyte removal from the circulation by the spleen, and endothelial cells. Controls with preimmune serum were nega- erythrocyte binding to the vascular endothelium (13-15). The tive. This antibody interaction was inhibited by murine eryth- glycosylated ectodomains of the glycophorins form blood- rocyte ghosts and purified glycophorins. Our past work iden- group antigens, bind lectins, and act as receptors for bacteria tified several endothelial surface sialoglycoproteins including a and viruses (13, 14). The development of autoantibodies to 60-kDa glycoprotein (gp6O) that (i) interacts with albumin, (ii) their ectodomains may result in autoimmune hemolytic ane- binds Limax flavus, Ricinus communis, and Triticum vulgare mia (16). Their endodomains interact with the infrastructure agglutinins but not other lectins, (iii) is sequentially precip- of the plasmalemma via a high-affinity linkage protein, band itated from '25I-labeled cell lysates by using R. communis 4.1 (17, 18), which ultimately may modulate changes in agglutinin followed by T. vulgare agglutinin, and (iv) is sensitive red-cell rigidity and deformability upon interactions at the to sialidase digestion. Immunoblotting ofsuch precipitates with cell surface (18, 19). agp demonstrates that lectins recognize the same glycoprotein, Murine glycophorins, gp2 and gp3, like their human coun- namely gp6O. These results indicate that gp6O, a major endo- terparts, glycophorins A and B, are major sialoglycoproteins thelial surface sialoglycoprotein, shares antigenic epitope(s) of the erythrocyte membrane (13, 14) and, until recently, with glycophorin. have been considered to be erythroid cell-specific. Based on indirect evidence, the presence ofglycophorin-like molecules in a variety of nonerythroid cells has been suggested for the The highly sialylated, polyanionic glycocalyx of the micro- glycophorins A, B, and C (20-26). In this study, we inves- vascular endothelium creates a significant permselective tigate possible structural similarities between endothelial barrier that restricts intravascular flow and transvascular sialoglycoproteins and glycophorins by using a polyclonal transport of the molecular and cellular constituents of the antiserum directed against murine glycophorins. Preliminary blood. The negative charge of most plasma macromolecules, results ofthis work have been published in abstract form (27). ofall circulating blood cells, and ofthe endothelial glycocalyx ensures significant electrorepulsion between these elements, thereby limiting nonspecific contact between the vascular METHODS wall and both the blood cells and the plasma proteins (for Cell Cultures. Microvascular endothelial cells from rat details, see ref. 1). Little is known about the glycoproteins epididymal fat pads [rat fat capillary (RFC) cells] were that form the endothelial glycocalyx. Recently, we have isolated, grown, and plated to achieve confluent monolayers identified on the endothelial surface a group of sialoglyco- as described (1). Bovine endothelial cells isolated from the proteins (gpl4O, gpl20, gplOO, gp6O, and gp47). Some of pulmonary artery (BPA), pulmonary vein (BPV), and mi- these proteins may be involved in a number of important crovessels of lung tissue (BLMV) were generously provided vascular phenomena, including receptor-mediated transcy- by P. DelVecchio and were grown on plastic dishes in tosis, cellular diapedesis, hemostasis, and blood-borne me- Dulbecco's modified Eagle's medium (DMEM) containing tastasis (1). The specific binding ofalbumin to the endothelial 10% fetal bovine serum. The endothelial origin of the cell surface (2-5) via one of thdse sialoglycoproteins, gp6O (6), as monolayers was checked periodically (5). Murine erythro- well as two other albumin-binding proteins (7, 8), apparently leukemia (MEL) cells were grown and induced to differen- increases capillary wall permselectivity (9) by increasing both tiate as described (28). charge and volume exclusion within the endothelial glyco- calyx (10-12). Abbreviations: agp, anti-glycophorin; BPA, bovine pulmonary ar- In contrast to endothelial sialoglycoproteins, the main tery; BPV, bovine pulmonary vein; BLMV, bovine lung microvessel; sialoglycoproteins of erythrocytes, collectively known as RFC, rat fat capillary; MEL, murine erythroleukemia; RCA, Ricinus glycophorins, have been investigated extensively (for re- communis agglutinin; WGA, Triticum vulgare (wheat germ) agglu- view, see refs. 13 and 14) and have served as a benchmark for tinin. *Present address: University ofCalifornia at San Diego, Cellular and Molecular Medicine, M-051, La Jolla, CA 92093. The publication costs of this article were defrayed in part by page charge tPresent address: Merck Sharp & Dohme Research Laboratories, payment. This article must therefore be hereby marked "advertisement" Department of Cancer Research, Building 16-3, West Point, PA in accordance with 18 U.S.C. §1734 solely to indicate this fact. 19486. 6843 Downloaded by guest on September 30, 2021 6844 Cell Biology: Schnitzer et al. Proc. Natl. Acad Sci. USA 87 (1990) Immunoprecipitation of Radioiodinated Cell Surface Poly- PAGE and electrotransferred onto filters. Endothelial cells peptides. As described previously (1), confluent monolayers derived from the microvasculature of the rat epididymal fat of endothelial cells were radioiodinated by the lactoperox- pad (RFC) and bovine lung (BLMV) and from large vessels idase technique and processed for immunoaffinity chroma- such as bovine pulmonary artery (BPA) and vein (BPV) were tography and SDS/PAGE. MEL cells (107 per sample) were used. Immunoblotting with agp specifically identified a single washed several times in phosphate-buffered saline (PBS), 60-kDa polypeptide in each cell line tested (Fig. 1). Controls pelleted, and then resuspended in 1.0 ml ofPBS containing75 with preimmune and nonimmune serum were negative. Since gg of lactoperoxidase and 50 ng (1.0 mCi; 37 MBq) of Na125I each confluent endothelial cell monolayer was processed (Amersham). Initially and at four successive 3-min intervals, under identical conditions and the same lysate volume was 10 Al of0.35 mM H202 in double-distilled water was added to loaded onto each gel lane, the stronger signal observed'for the the cell suspension and''mixed by mild circular agitation (5 endothelial cells derived from microvessels (RFC and sec). After 15 min, the reaction was stopped by first pelleting BLMV) rather than large vessels (BPA and BPV) suggests the cells, then aspirating the reaction mixture, and'finally that microvascular endothelium expresses more of this pro- washing the cells with three 1-ml portions of PBS (1 min per tein per unit surface area than other endothelia. Furthermore, wash). The cells were lysed and processed for immunoaftin- microvascular cells appear to express more gp6O on a per cell ity chromatography and SDS/PAGE as described (28). basis than other endothelia, based on the observation that Radioiodination of IgG Fraction. IgG, purified from anti- BPA and BLMV cells have similar cell surface densitie's, glycophorin (agp) serum by using protein A-Sepharose beads while BPV cells are much less spread out and have a greater as per manufacturer's instructions (Pharmacia), was radio- cell surface density (data not shown). iodinated in 0.5 ml ofPBS (0.5 mg/ml) with 0.2 mg ofiodogen Immunoprecipitation of Radioiodinated Plasmalemmal Pro- (1,4,5,6-tetrachloro-3a,6a-diphenylglycouril) -and 1.5 mCi of teins with agp Serum. Lactoperoxidase-catalyzed radioiodi- Na125I stock (75 ng) as described (5). Free 1251 was removed nation of intact cells, followed by cell lysis and lysate by usinga 10-ml Bio-Gel P-6 desalting column (Bio-Rad). The processing through SDS/PAGE and autoradiography,'dem- specific activity was -2.5 mCi/mg and the purity of the onstrated the presence ofseveral cell surface proteins. Immu- labeled protein was verified by SDS/PAGE. noprecipitation ofthis radiolabeled