Isolation, physicochemical properties, and the macromolecular composition of the vitelline and fertilization envelopes from Xenopus laevis eggs

As a step toward defining in molecular terms the sperm-triggered block to polyspermy reaction established by the egg at fertilization, vitelline (VE) and fertilization (FE) envelopes were isolated from eggs of the Sounth African clawed toad Xenopus laevis and some of their physicochemical properties determined. Envelopes were isolated after lysis of the fertilized or unfertilized eggs by sieving techniques; isolated envelopes retained their in situ morphology as determined by electron microscopy. The isolated envelopes had different solubility properties and, in general, VE was more readily dissolved by aqueous solvents than FE, although both could be completely dissolved by detergents or chaotropic agents. Changes in envelope solubility correlated with the progression of the cortical reaction implicating a role for cortical granule material in modifying the solubility properties of the envelope. The VE and FE were composed of protein and carbohydrate with no lipid components detected. As determined by immunodiffusion experiments, the FE contained the same antigens as the VE plus components derived from the cortical granules and the innermost jelly layer, J. The macromolecular composition of the envelopes was determined by sodium dodecyl sulfate gel electrophoresis. The VE contained at least 11 glycoproteins with molecular weights ranging from 125 000 to less than 16 000 with two components (40 000 and 33 000) accounting for almost two-thirds of the total stainable material. The FE contained ten glycoproteins that had the same molecular weights as those in the VE. One glycoprotein component underwent a reduction in molecular weight from 77 000 to 67 500 when the VE was converted to the FE. This molecular weight change was interpreted as the probable result of limited proteolysis. In addition, the FE gel electrophoresis patterns contained macromolecular components derived from the cortical granules and jelly layer, J, consistent with the immunodiffusion experiments. These components were absent when the FE was prepared in the absence of Ca2+, suggesting a role for Ca2+ in binding the VE, cortical granules, and J components together. We concluded that the conversion of the glycoproteinaceous VE to FE at fertilization is caused by interaction of the VE with components from the cortical granules and jelly layer J. These interactions are of both a chemical and physical nature.     

Immunoelectrophoretic analysis of the porcine zona pellucida

Antigens of the porcine zona pellucida were evaluated by 2-dimensional and line immunoelectrophoretic techniques. Zona antigen preparations studied included heat-solubilized isolated zonae pellucidae (SIZP), a purified 60 000 Mr glycoprotein (PPZA, purified pig zona antigen), and two fractions of this 60 000 Mr zona component which had been exposed to SDS (ZP3-E1C and ZP3-E2C). Antisera were raised to intact zonae pellucidae (IZP), SIZP and PPZA. Collectively, electrophoretic data revealed that the porcine zona system is antigenically complex with each zona antiserum tested detecting numerous antigens in the various zona preparations. These antigens, however, all had similar electrophoretic mobilities, and this limited the resolution of these techniques. The 60 000 Mr pig zona macromolecule (ZP3) appeared to be the most immunogenic of the three major pig zona glycoproteins since antisera prepared against IZP or SIZP reacted primarily with this component. However, the 60 000 Mr component does share antigenic determinants with the other major zona glycoproteins as revealed by cross-reactions of the antisera with the various zona preparations. Electrophoretic studies also suggested that the various zona antisera could distinguish, with different degrees of sensitivity, multiple antigenic determinants on the individual zona macromolecules. These studies also indicated that SDS treatment of zona glycoproteins does alter the antigenicity of the macromolecule, both with respect to the total number and individual identity of antigens detected.     

Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.     

Lectins as probes of chromatin structure. Binding of concanavalin A to purified rat liver chromatin

Concanavalin A (Con A) binds specifically to rat liver chromatin. The extent of binding is directly proportional to both chromatin and concanavalin A concentration. It is reversible and inhibited by specific sugars for which concanavalin A has a binding site. Scatchard analysis reveals the presence of one type of Con A-binding site, with an apparent dissociation constant of 3 X 10(-7) M. A maximum of 10 pmol of Con A binds to 10 microgram of chromatin, indicating an average of one binding site/1400 base pairs of DNA. To identify the polypeptide chains which contain Con A-binding sites, chromosomal proteins were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. Con A receptors were localized by incubating the gel in 125I-Con A and subsequent autoradiography. Three major polypeptide bands which bind Con A were identified among the nonhistone chromosomal proteins. The apparent molecular weights of these glycoproteins are 135,000, 125,000, and 69,000. We suggest that lectins may serve as probes for the study of the organization of specific components in chromatin.     

Proteins and glycoproteins of membranes from developing chick red cells

Membrane vesicles produced when chick erythroid cells are disrupted by nitrogen cavitation were isolated by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Sodium dodecyl sulfate-polyacrylamide gel analysis of isolated membranes shows eight to ten proteins and four to five glycoproteins. Membranes must be prepared with protease inhibitors, otherwise an endogenous activity degrades high molecular weight polypeptide components. Red cells from several stages of development (5- and 17-day embryos and adult chickens) all appear to have the same major embrane proteins. However, primitive erythroid cells from 5-day embryos lack a Mr = 40,000 glycoprotein that is present in definitive erythrocytes from 17-day embryos and from adult chickens; erythrocytes from young chicks show a decrease in the amount of a glycoprotein of Mr = 50,000. Lactoperoxidase-catalyzed iodination of intact 5-day embryonic red cells detects three surface components which comigrate with the membrane glycoproteins on sodium dodecyl sulfate polyacrylamide gels.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Concanavalin A binding to the epithelial surface of the developing mouse olfactory placode

Concanavalin A (Con A), a lectin binding to mannosyl and glucosyl residues of glycoproteins and glycolipids, was used to study the appearance of carbohydrate-rich cell surface material on the olfactory placode and nasal processes which contribute to formidine was also used in an attempt to correlate changes in labeling index with formation of the olfactory placode and nasal processes. The cell surface of the early frontonasal epithelium binds Con A very little, if at all, but Con A binding was observed when the olfactory placode could be identified as a plate of cuboidal cells that exhibited a reduced labeling index. During the period of formation of the nasal processes, Con A binding was observed on the facial epithelium including the presumptive contact region. There was also a decline in the labeling index throughout primary palate formation. This study provides three criteria by which the olfactory placode can be identified: a morphological change of placode cells to a cuboidal shape, a synthesis or rearrangement of surface coat material that binds Con A, and a reduced labeling index.     

Immunoreactivity of glycoprotein IIb is present in metastasized but not in non-metastasized primary malignant melanoma

Glycoprotein IIb-IIIa (integrin alpha IIb beta3) is an adhesive receptor involved in platelet aggregation and adhesion to the extracellular matrix. Previous studies showed the presence of IIb-IIIa-like glycoproteins on cells of melanoma cell lines and on cells of lymph node metastases. This study evaluates the presence of glycoprotein IIb-IIIa subunits on cells of primary cutaneous malignant melanomas with (n = 4) and without (n = 9) metastases over a period of 6 years and on naevus cells (n = 4). Monoclonal antibodies directed against the subunits of the glycoprotein IIb-IIIa receptor were used on paraffin-embedded sections and evaluated by means of immunohistochemistry. The glycoprotein IIb subunit was exclusively present on cells of metastatic melanomas. It was not found on non-metastatic melanomas or benign melanocytes. These data favour the role of the integrin receptor glycoprotein IIb-IIIa in the metastatic behaviour of malignant melanomas.     

In vitro viability of cryopreserved equine embryos following different freezing protocols

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4 degrees C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterisation of peanut agglutinin-binding glycoproteins from Eimeria tenella

A group of glycoproteins, which strongly bind peanut agglutinin (PNA) was found in Eimeria tenella. Two major antigenic glycoproteins, Et110gp and Et35gp, were identified in sporulated oocysts and sporozoites. Molecular characterisation of carbohydrate moieties (lectin binding, enzymic hydrolysis and monosaccharide composition) revealed that both glycoproteins are rich in galactose and N-acetylgalactosamine, and appear to be sialylated. Both glycoproteins were susceptible to treatment with neuraminidase followed by O-glycosidase, suggesting that the oligosaccharide chains are attached to the protein by an O-glycosidic linkage to serine and/or threonine. Purified Et35gp contained a large number of serine (14) and threonine (33) residues, and was rich in glycine. This protein aggregated after repetitive lyophilisation and migrated on SDS-PAGE gels as an 85,000 protein. Sera against purified Et35gp raised in chickens and rabbits, and anti-E. tenella immune chicken serum recognised both antigens on blots and on the surface of sporozoites. Chickens immunised with purified Et35gp were not protected against coccidial infection.     

Identification and partial characterization of a glycoprotein species with high affinity for methylmercury in peripheral nervous tissues of man and experimental animals

A small amount of a glycoprotein species (21-kDa glycoprotein) with high affinity for methylmercury (MeHg) was detected in the post-nuclear or post-mitochondrial supernatant fraction of the homogenate of rat sciatic nerve on electrophoresis and autoradiography after binding of Me203Hg to the fraction. The 21-kDa glycoprotein was also found in the subcellular fractions of mouse, hamster, guinea pig, rabbit and human peripheral nervous tissues. Experiments with the cellular fractions of the tissues revealed that the 21-kDa glycoprotein is localized mainly in the myelin fraction, whereas it was not found in the cellular fractions of brain, spinal cord and nonneural tissues, such as kidney and liver. The specific binding activity of the 21-kDa glycoprotein with MeHg was 12-15 fold that of the major myelin protein, Po. It was shown that the interaction of the 21-kDa glycoprotein with MeHg was mediated through sulfhydryl groups in experiments with iodoacetamide and dithiothreitol. The amino acid compositions of the rat and human 21-kDa glycoproteins were similar but very different from that of a typical metallothionein. The N-terminal amino acid sequences of the two components of the rat 21-kDa glycoprotein were identical to those of P0 and PMP-22, respectively. The in vitro binding of MeHg was also observed in the myelin fraction obtained from the sciatic nerves of MeHg-dosed rats.     

Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.     

Role of surface glycoproteins in human platelet function

Glycoproteins present at the external surface of cells probably play specific roles in cellular function. Increasing evidence suggests that the glycoproteins span the plasma membrane with the bulk of the bound carbohydrate asymmetrically distributed on the outer surface. Micellar association of glycoproteins in membranes leads to pore formation and functional roles in transport through the membrane, while surface glycoproteins have been shown to be enzymes, to determine cell specificity and contribute to the cell surface change. The platelet plasma membrane contains 3 major glycoproteins, glycoproteins I, II and III as characterized in order of their decreasing molecular weight. Glycoprotein I appears to have the highest sialic acid content and to give rise to a platelet specific acidic macroglycopeptide on trypsin digestion. Specific glycoprotein abnormalities in the platelets of patients with Glanzmann's thrombasthenia suggest that the glycoproteins play a role in the mechanism of platelet aggregation. A much reduced content of glycoprotein I in the platelets of 2 patients with the Bernard Soulier syndrome may be associated with their defective adhesion to subendothelium and indicates a possible relationship on the platelet surface with the von Willebrand factor protein. Preliminary evidence suggests that in common with other plasma membranes the platelet membrane has a fluid structure and that the organization of the glycoproteins on the platelet surface is extremely sensitive to stimuli and susceptible to change.     

Functional and structural identification of a new lectin activity of Borrelia recurrentis spirochetes

1. Haemagglutinating activity (HA) was found and characterized in lysate of the spirochete Borrelia recurrentis. 2. The highest HA was observed using native rabbit red blood cells (RBC), especially oxidized rabbit RBC. 3. In a haemagglutination inhibition test the HA showed an affinity with monosaccharides D-glucosamine, D-galactosamine and N-acetyl-D-mannosamine and several glycoproteins and polysaccharides. 4. An inhibitory effect was also achieved by mouse monoclonal antibody H 9724, polyclonal antibodies from B. recurrentis infected mice and with rabbit polyclonal antibodies directed against B. recurrentis HA. 5. B. recurrentis proteins components of 29, 33, 41 and 55 kDa were identified by immunoblotting as structural subunits of complex binding activity of a new agglutinin.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Production and characterization of bovine herpesvirus 1 glycoprotein B ectodomain derivatives in an hsp70A gene promoter-based expression system

Different derivatives of bovine herpesvirus 1 (BHV-1) glycoprotein B (gB) ectodomain were expressed in a novel heat-shock expression system. The putative ectodomain, gBt, and the N-terminal subunit, gBb, were of the expected molecular weight and were secreted. Their production were heat-inducible and the purified proteins were able to elicit antibody responses in mice of a comparable level as induced by authentic gB. The truncated C-terminal subunit, gBct, was retained in the endoplasmic reticulum. Our studies suggest that the gBb subunit may play a major role in constituting the overall configuration of gB and is required for the intracellular transport of gB.     

Demonstration of discrete secreted and membrane-bound ocular mucins in the dog

Our aims were to separate and characterize secreted canine ocular mucins, and to provide definitive evidence of membrane-bound mucins at the canine ocular surface. Mucus was collected by suction from the ocular surface of normal dogs and dispersed in guanidine hydrochloride and a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation separated secreted mucins from membranes, which were collected from the top of the gradients. Membranes were extracted with octyl glucoside and screened using lectins and anti-mucin antibodies. Gradient fractions containing secreted mucins were constituted into pools on the basis of differential lectin and antibody staining. High molecular weight material from each pool was purified by gel filtration. This material, and the membrane extract, were reduced and alkylated. Vacuum blotting of separated materials after agarose gel electrophoresis was used to compare subunit structure. Density gradient profiles indicated three principal secreted glycoprotein peaks: one staining strongly with anti-mucin antibodies. Gel filtration demonstrated that each contained high molecular weight material. Vacuum blots demonstrated the presence of two secreted glycoproteins with differently sized subunits. On the basis of buoyant density, one of these may be lipid complexed. Membrane extracted material stained with anti-mucin antibodies, and vacuum blotting of this material provided evidence for two membrane-bound components. In conclusion, we have shown that normal canine ocular mucus contains two secreted mucins, each exhibiting different subunit structure; one of these mucins may undergo lipid complexation. Normal canine ocular mucus also contains two membrane-bound mucins: one of which is unique among membrane mucins in showing subunit structure.     

Fine structural and functional consequences of deglycosylation of the platelet adhesion receptor GPIb-IX (CD 42b)

To investigate the role of the glycosylation of the platelet receptor glycoprotein Ib (GPIb, CD 42b), platelets and purified GPIb were deglycosylated by neuraminidase, O- and N-glycosidases. N-deglycosylation and neuraminic-acid cleavage had little effect on ristocetin and botrocetin-induced platelet agglutination. However, O-deglycosylation reduced the response by approximately 50%, and total deglycosylation (the combination of all three glycosidases) fully abolished the response to ristocetin. Interestingly, binding of von Willebrand Factor (vWF) to purified GPIb in the presence of ristocetin and botrocetin in a standardized microtiter plate assay was not altered by partial or even by total deglycosylation. Electron microscopy indicated that the normally stretched approximately 50 nm long molecule was approximately 32 nm after N-deglycosylation, approximately 20 nm after O-deglycosylation, and reduced in a approximately 15 nm long collapse by total deglycosylation. These results suggest that deglycosylation has major structural impacts on GPIb, strongly impairing platelet-vWF interactions; however, vWF binding to isolated GPIb remains unaffected.     

P-selectin mediates adhesion of the human melanoma cell line NKI-4: identification of glycoprotein ligands

Activated endothelial cells and stimulated platelets express the cell adhesion molecule P-selectin (CD62P), which mediates adhesion to various leukocytes and certain types of cancer cells. In this study, we show Ca2+-dependent binding of P-selectin to NKI-4 cells, a cell line derived from a human melanoma. The binding is inhibited by P7 (a leukocyte adhesion blocking mAb against P-selectin), but not by PL5 (a leukocyte adhesion blocking mAb against P-selectin glycoprotein ligand-1; PSGL-1). Further, expression of PSGL-1 could not be detected on NKI-4 cells by either PL5 mAb or an Ab against a synthetic peptide corresponding to a portion of the PSGL-1 sequence. P-selectin affinity chromatography of lysates from in vivo [3H]-glucosamine-labeled NKI-4 cells resulted in the isolation of three glycoproteins, with apparent molecular masses of approximately 250, approximately 110, and approximately 100 kDa under reducing conditions and approximately 230, approximately 105, and approximately 85 kDa under nonreducing conditions. These molecules could be precipitated by P-selectin, but not by E-selectin. EDTA and the P7 mAb, but not the PL5 mAb, inhibited the binding of P-selectin to the purified ligands. Surprisingly, we found that sodium chlorate, a sulfation inhibitor, did not inhibit the binding of P-selectin to NKI-4 cells and that [35S]-sulfate did not label the NKI-4 cell ligands. We conclude that P-selectin-dependent adhesion of the human melanoma cell line NKI-4 is mediated by a novel class of glycoprotein ligands.