Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.     

Subunit structure of thyrotropin receptors expressed on the cell surface

We studied cell surface thyrotropin receptor (TSHR) by biotinylating proteins on the surface of metabolically labeled, intact cells. In addition to TSHR cleaved into A and B subunits, mature single-chain receptors with complex carbohydrate were also present on the cell surface. A low A/B subunit ratio indicated partial shedding of extracellular A subunits from transmembrane B subunits. TSHR cleavage at upstream site 1 (within amino acid residues 305-316) would generate a B subunit of 51-52 kDa. However, only smaller B subunits (40-46 kDa) were detected, corresponding to N termini from residues approximately 370 (site 2) extending downstream to the region of B subunit insertion into the plasma membrane. The intervening C peptide region between sites 1 and 2 could not be purified from TSHR epitope-tagged (c-myc) within this region. However, the small proportion of B subunits recovered with a c-myc antibody were larger (45-52 kDa) than the majority of B subunits recovered with a C-terminal antibody. In conclusion, our study provides the first characterization of cell surface TSHR including their A and B subunits. Single-chain, mature TSHR do exist on the cell surface. The C peptide lost during intramolecular cleavage disintegrates rapidly following cleavage at upstream site 1 of the single-chain TSHR into A and B subunits. N-terminal disintegration of the B subunit pauses at site 2, but then progresses downstream to the vicinity of the plasma membrane, revealing a novel mechanism for A subunit shedding.     

Carbohydrate-mediated cell adhesion to glycoproteins immobilized on nitrocellulose

A method that allows the estimation of carbohydrate-mediated cell adhesion to glycoproteins and polysaccharides immobilized to a nitrocellulose matrix is described. Specificity of adhesion by indicator cells (Chang liver) has been verified using glycoconjugates with defined carbohydrate structure. Two independent receptor systems with beta-galactose or alpha-fucose specificity, respectively, have been demonstrated by this method to occur on Chang liver cells. The method is also applicable for other indicator cells like murine fibrosarcoma cells and has been used for the analysis of dot-blots and Western blots of glycoproteins.     

Multiple carbohydrate receptors on lymphocytes revealed by adhesion to immobilized polysaccharides

Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.     

Syndecans play dual roles as cell adhesion receptors and docking receptors

Syndecan are a family of cell surface heparan sulfate proteoglycans that act as cell surface receptors. Most cell surface receptors have a limited number and type of ligand interactions, responding only to the binding of (a) specific ligand(s). In contrast, syndecans can interact with various numbers and types of ligands, and thus play more diverse roles than others. Various syndecan functions have not yet been fully classified and categorized, but we herein review previous studies suggesting that syndecans play dual function as cell surface receptors by acting as both adhesion receptors and docking receptors. Through this dual regulatory function, syndecans are capable of regulating both intra- and extracellular activities, potentially altering a variety of cell behaviors.     

Cross-linking of cell surface receptors enhances cooperativity of molecular adhesion

Cooperativity of molecular adhesion has been proposed as a mechanism for enhanced binding strength of adhesion molecules on the cell surface. Direct evidence for its mechanism, however, has been lacking until now. Atomic force microscopy (AFM) was used to measure the adhesive strength between concanavalin A (Con A) coupled to an AFM tip and Con A receptors on the surface of NIH3T3 fibroblast cells. Cross-linking of receptors with either glutaraldehyde or 3, 3'-dithio-bis(sulfosuccinimidylproprionate) (DTSSP) led to an increase in adhesion that could be attributed to enhanced cooperativity among adhesion complexes. An increase in loading rate due to greater stiffness of fixed cells also contributed to the twofold increase in binding strength. These results show that receptor cross-linking can greatly contribute to a total increase in cell adhesion by creating a shift toward cooperative binding of receptors.     

Concanavalin A surface receptors and cytoplasmic actin in cell adhesion.

A double immunofluorescence staining technique to locate concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell was applied to monolayer cultures of rat foetal fibroblasts during cell detachment induced by trypsin and during cell attachment to glass substratum. Con A receptors were demonstrated by fluorescein-isothiocyanate-labelled Con A (FITC-Con A) and actin by specific anti-actin antibody (AAA) traced with rhodamine-labelled goat anti-human globulin (R-AHG). Untreated, control cells had an elongated shape, Con A receptors restricted to cell margins and prominent actin filaments. After 2 min treatment with 0.001% trypsin the cells became angular with Con A receptors in clusters and actin in a diffuse or aggreagate staining pattern. Progressive cell rounding followed and this was accompanied by the development of long, thin, arborized cell processes, studded with Con A receptors and containing fine actin filaments. Complete cell rounding preceded cell detachment. The sites of detached cells were marked by fine aggregates containing Con A receptors and actin. In cells attaching to a glass substratum, actin was present in a diffusely stained or aggregate pattern in round cells, in filaments restricted to cell margins in partially spread cells and in numerous filaments in fully spread cells. Con A receptors were present in clusters in round cells, in clusters or caps in partially spread cells and in cell margins in fully spread cells. Binding of FITC-Con A to partially spread cells resulted in dissolution of the few, newly formed, actin filaments. We believe our observations are consistent with the idea that actin filaments, formed during cell attachment, contribute towards the maintenance of cell adhesion by helping in the preservation of cell shape and by anchorage of Con A receptors at points of cell attachment to the substratum.     

Bioactive microarrays immobilized on low-fouling surfaces to study specific endothelial cell adhesion

With the aim to study how to modulate the specific endothelial cell patterning and responses on biomaterials surfaces, bioactive microarrays were developed and validated for specific cell patterning. These microarrays were made of low-fouling surfaces, that prevent nonspecific cell adhesion, bearing bioactive molecules at given known locations by presenting specific ligands to cell receptors. Arrays of bioactive molecules (RGD, REDV, and SVVYGLR sequences and vascular endothelial growth factor (VEGF)) were immobilized on a carboxy-methyl-dextran low-fouling surface and were exposed to human endothelial cells and fibroblasts to screen for the effect of bioactive spot molecular composition on cell adhesion. Endothelial cells only were sensitive to RGD peptide co-immobilized with REDV or SVVYGLR sequences: they induced a reduction in cell spreading and a loss of actin stress fibers. RGD co-immobilized with VEGF also resulted in the reorganization of actin filaments and focal points in endothelial cells. Combination of RGD with these endothelial cell-selective biomolecules did not elicit a strong adhesion phenotype but rather one characteristic of migrating cells.     

Oligosaccharide side chains on human secretory IgA serve as receptors for ricin

Secretory IgA (sIgA) Abs are polymeric Igs comprised of two or more IgA monomers joined together at their C termini and covalently associated with a 70-kDa glycoprotein called secretory component. As the predominant Ig type in gastrointestinal sections, sIgA Abs are centrally important in adaptive immunity to enteropathogenic bacteria, viruses, and toxins. In this study, we demonstrate that sIgA Abs may also function in innate defense against ricin, a naturally occurring, galactose-specific plant lectin with extremely potent shiga toxin-like enzymatic activity. In lectin blot overlay assays, we found that ricin bound to secretory component and the H chain of human IgA, and this binding was inhibited by the addition of excess galactose. The toxin also recognized IgM (albeit with less affinity than to IgA), but not IgG. Ricin bound to both human IgA1 and IgA2, primarily via N-linked oligosaccharide side chains. At 100-fold molar excess concentration, sIgA (but not IgG) Abs inhibited ricin attachment to the apical surfaces of polarized intestinal epithelial cells grown in culture. sIgA Abs also visibly reduced toxin binding to the luminal surfaces of human duodenum in tissue section overlay assays. We conclude that sIgA Abs in mucosal secretions may serve as receptor analogues for ricin, thereby reducing the effective dose of toxin capable of gaining access to glycolipid and glycoprotein receptors on epithelial cell surfaces.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Paradigms for glycan-binding receptors in cell adhesion

Diverse glycans found on the surfaces of mammalian cells provide a basis for selective adhesion between cells mediated by glycan-specific receptors. Well-understood examples of cell adhesion based on such interactions include selectin-mediated rolling of leukocytes on endothelia. Other receptors with similar selectivity for specific sugar epitopes on cell surfaces are being characterised. However, the simple paradigm of adhesion resulting from receptors on one cell binding to glycans on another cell applies in only a limited number of systems. Instead, glycans and receptor-glycan interactions often modulate adhesion in indirect ways, such as by changing the organisation of cell surface glycoproteins and by antagonising the effect of protein adhesion systems.     

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.     

Anti-idiotypic antibodies as probes of cell surface receptors

Summary Anti-idiotypic antibodies have proven to have unique applications as probes in both functional and biochemical studies of cell surface receptors. Anti-idiotypic receptor antibodies have been prepared to antibodies which bind to purified ligand, as in the case of insulin, retinol-binding protein, the mammalian reovirus receptor, and the neutrophil chemotatic receptor, and to natural ligand analogs, such as the beta-adrenergic antagonist alprenolol. These systems have documented the usefulness of anti-idiotypic antibodies in the quantitation and modulation of specific membrane receptors on a variety of cell types. Anti-idotypic antibodies have also been utilized for the isolation of specific membrane receptors, e.g., reovirus and B-1 H globulin receptors. Some anti-idiotypic receptor antibodies, e.g., insulin and reovirus systems, have been shown to mimic the physiological properties of ligand upon binding to cellular receptors. These antibodies enable a new dimension of both receptor based cellular studies and therapeutic regimens. This review focuses on the past use of anti-idiotypic antibodies as probes of cell surface receptors, and on the methodologies required for the successful application of anti-idiotypic antibodies for use in further membrane receptor studies, and of the genes which encode and regulate these receptors. We also discuss the use of anti-idiotypic antibodies in the understanding of and therapeutic approach to receptor related diseases.Dr. Gaulton received the NRSA Viral Oncology Training Grant t# CA 09031.     

Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages

Macrophages are thought to represent one of the first cell types in the body to be infected during the early stage of human immunodeficiency virus type 1 (HIV-1) transmission and represent a potential viral reservoir in vivo. Thus, an understanding of HIV-1 attachment to these cells is fundamental to the development of novel anti-HIV-1 therapies. Although one of the major targets of HIV-1 in vivo--CD4(+) T lymphocytes--express high CD4 levels, other major targets such as macrophages do not. We asked in this study whether this low CD4 level on macrophages is sufficient to support HIV-1 attachment to these cells or whether cell surface proteins other than CD4 are required for this process. We show that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to macrophages. Importantly, we find that heparan sulfate proteoglycans (HSPGs) serve as the main class of attachment receptors for HIV-1 on macrophages. Most importantly, we demonstrate that a single family of HSPGs, the syndecans, efficiently mediates HIV-1 attachment and represents an abundant class of attachment receptors on macrophages.     

Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.     

Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome

The selective recognition of ubiquitin conjugates by proteasomes is a key step in protein degradation. The receptors that mediate this step have yet to be clearly defined although specific candidates exist. Here we show that the proteasome directly recognizes ubiquitin chains through a specific subunit, Rpn10, and also recognizes chains indirectly through Rad23, a reversibly bound proteasome cofactor. Both binding events can be observed in purified biochemical systems. A block substitution in the chain-binding ubiquitin interacting motif of RPN10 when combined with a null mutation in RAD23 results in a synthetic defect in protein degradation consistent with the view that the direct and indirect recognition modes function to some extent redundantly in vivo. Rad23 and the deubiquitinating enzyme Ubp6 both bind proteasome subunit Rpn1 through N-terminal ubiquitin-like domains. Surprisingly, Rad23 and Ubp6 do not compete with each other for proteasome binding. Thus, Rpn1 may act as a scaffold to assemble on the proteasome multiple proteins that act to either bind or hydrolyze multiubiquitin chains.     

A quantitative microassay of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on polystyrene plates.

A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).     

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).     

G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils

Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection. leukocyte; constitutive activity; mechanotransduction; formyl peptide receptor