Domain organization of Mac-2 binding protein and its oligomerization to linear and ring-like structures.

The multidomain Mac-2 binding protein (M2BP) is present in serum and in the extracellular matrix in the form of linear and ring-shaped oligomers, which interact with galectin-3, fibronectin, collagens, integrins and other large glycoproteins. Domain 1 of M2BP (M2BP-1) shows homology with the cysteine-rich SRCR domain of scavanger receptor. Domains 2 and 3 are related to the dimerization domains BTB/POZ and IVR of the Drosophila kelch protein. Recombinant M2BP, its N-terminal domain M2BP-1 and a fragment consisting of putative domains 2, 3 and 4 (M2BP-2,3,4) were investigated by scanning transmission electron microscopy, transmission electron microscopy, analytical ultracentrifugation and binding assays. The ring oligomers formed by the intact protein are comprised of approximately 14 nm long segments composed of two 92 kDa M2BP monomers. Although the rings vary in size, decamers predominate. The various linear oligomers also observed are probably ring precursors, dimers predominate. M2BP-1 exhibits a native fold, does not oligomerize and is inactive in cell attachment. M2BP-2,3,4 aggregates to heterogeneous, protein filled ring-like structures as shown by metal shadowed preparations. These aggregates retain the cell-adhesive potential indicating native folding. It is hypothesized that the rings provide an interaction pattern for multivalent interactions of M2BP with target molecules or complexes of ligands.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.     

Functional studies on recombinant domains of Mac-2-binding protein

Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP. Both fragments were obtained in a native and glycosylated form, as analyzed by CD spectroscopy, trypsin susceptibility, and enzymatic deglycosylation. These results strongly suggest that both fragments are autonomous folding units. All three potential N-glycosylation sites in M2BP-1,2 and all four in M2BP-3,4 were found to be occupied. M2BP-1,2 expressed in tunicamycin-treated cells contained no glycosyl residues, indicating that O-glycosylation is not occurring. Ultracentrifugation revealed that M2BP-1,2 is homogeneously dimeric in the nanomolar range reflecting the properties of intact M2BP. Domain 2 (BTB/POZ domain) is thus identified as the dimerization domain of M2BP, because it has been formerly shown that recombinant domain 1 is monomeric. M2BP-3,4 showed a concentration-dependent self-association, and aggregates of different size and shape were shown by electron microscopy. In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2-4 still has the ability to form ring-like structures, although the rings are protein-filled, and thus domain 2 appears to be indispensable for ring formation. Solid phase assays showed that M2BP-3,4 contains binding sites for galectin-3, nidogen, and collagens V and VI, whereas M2BP-1,2 is inactive in binding. Both fragments showed no cell adhesive activity in contrast to native M2BP, suggesting that a concerted binding action and/or multivalent interactions of rings are necessary for cell attachment.     

A comparative study on the purification of the Amaranthus leucocarpus syn. hypocondriacus lectin.

Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.     

Folding and function of I domain-deleted Mac-1 and lymphocyte function-associated antigen-1

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between beta-sheets 2 and 3 of the predicted beta-propeller domain of the integrin alpha subunit. We deleted the I domain from the integrin alpha(M) and alpha(L) subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less alpha(M) and alpha(L) subunits were expressed in association with the wild-type beta(2) subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative beta-propeller and C-terminal regions of the alpha(M) and alpha(L) subunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the beta-propeller domain of the alpha(M) subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

Cellular localization and characterization of proteins that bind high density lipoprotein.

High density lipoprotein (HDL) stimulates excretion of excess intracellular cholesterol from cells, presumably by interacting with a cell-surface receptor. A 110 kDa membrane protein that is a candidate for the HDL receptor has been identified by ligand blot analysis. In this study we determined the cellular localization of this and other HDL-binding proteins and characterized their properties. The plasma membranes (PM) of cultured bovine aortic endothelial cells were labeled with trace amounts of [3H]cholesterol, and cell homogenates were fractionated on sucrose and Percoll gradients. Ligand blot analysis of homogenates of cultured bovine aortic endothelial cells demonstrated that cells contain multiple proteins that bind HDL3, including a major membrane protein with an apparent M(r) of 110 kDa and two minor ones with M(r) of 105 and 130 kDa. The gradient distribution of the 105, 110, and 130 kDa HDL-binding proteins mirrored that of labeled cholesterol and 5'-nucleotidase, both PM markers. Treatment of intact cells with the water-soluble cross-linker bis(sulfosuccinimidyl)suberate abolished the HDL binding activity of the 110 and 130 kDa proteins but not that of the 105 kDa protein. These findings suggest that the 105, 110, and 130 kDa HDL-binding proteins are localized to the PM and that at least two of these proteins are exposed to the extracellular fluid. Solubilized 110 and 130 kDa proteins were retained on wheat-germ agglutinin and abrin lectin columns, showing that they are glycoproteins. The cellular localization and physical properties of the 110 and 130 kDa proteins suggest that they may play a role in binding of HDL to the cell surface.     

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.     

Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.     

The insulin-like growth factor binding protein BP-25 is expressed by human breast cancer cells

Specific binding proteins are thought to modulate the effects of IGF-I. Previous work has demonstrated that media conditioned by human breast cancer cells contains IGF-I binding activity. Radiolabelled IGF-I incubated with serum-free conditioned media from the breast cancer cell line MDA-MB 231 eluted with an apparent M.W. of 35-40 kDa when analyzed by gel filtration chromatography at pH 7.4. The M.W. of this binding activity corresponded to that of BP-25, a binding protein cloned from the hepatocellular carcinoma cell line HepG2. Two breast cancer cell lines, MDA-MB 231 and Hs578T, were found to express BP-25 RNA. Specific BP-25 radioimmunoassay detected BP-25 production in the conditioned media of these two cell lines. Immunoprecipitation confirmed that metabolically labelled MDA-MB 231 released 30 kDa BP-25 into its medium. This study demonstrates that some breast cancer cells express the IGF-I binding protein, BP-25.     

Influence of quaternary structure on glycosylation. Differential subunit association affects the site-specific glycosylation of the common beta-chain from Mac-1 and LFA-1

The influence of quaternary structure on glycosylation was evaluated in a macrophage-like cell line, P388D1. This cell line simultaneously synthesizes two structurally related glycoproteins, Mac-1 and LFA-1. Mac-1 and LFA-1 each contain two subunits in noncovalent association in an alpha 1 beta 1 structure. The beta-chain polypeptides of these two glycoproteins have identical primary structures while their alpha-chain polypeptides are distinct. For both Mac-1 and LFA-1, the association of the alpha- and beta-chains occurs prior to any Golgi-mediated processing of the oligosaccharide moieties on either one of the subunits. To evaluate the effects of differential subunit association on the site-specific glycosylation of the beta-chain, [3H]glucosamine-labeled oligosaccharides were isolated from the beta-chain of Mac-1 and LFA-1 and were compared by a variety of enzymatic and chromatographic techniques. Reverse-phase high performance liquid chromatography analyses of tryptic-chymotryptic glycopeptides suggest that each beta-chain has at least five glycosylation sites. Structural analysis of oligosaccharides from each corresponding glycopeptide fraction of the beta-chains of Mac-1 or LFA-1 (comparing their glycosidase sensitivities, behavior on serial lectin affinity chromatography, size heterogeneity, extent of sialylation, and branching) indicates that the LFA-1 beta-chain is glycosylated substantially differently on at least four of its sites, compared to the corresponding sites of the Mac-1 beta-chain, even though they are simultaneously synthesized in the same cells. Thus, these data demonstrate that quaternary structure can influence the site-specific glycosylation of a protein, even when the polypeptide structure and the cellular glycosylation machinery remain constant.     

The macrophage alphaMbeta2 integrin alphaM lectin domain mediates the phagocytosis of chilled platelets

alpha(M)beta(2) integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands. Ligand binding occurs in the alpha(M) chain, which is composed of an I domain and a lectin domain. The alpha(M) I domain binds iC3b, fibrinogen, intercellular adhesion molecule-1, and other ligands and mediates the adhesion of neutrophils to platelet glycoprotein Ibalpha (GPIbalpha). alpha(M)beta(2) also recognizes beta-GlcNAc residues on GPIbalpha that are clustered on platelets after cooling. The phagocytosis of chilled platelets could be reconstituted when Chinese hamster ovary cells were transfected with alpha(M)beta(2). Replacement of the I domain or the lectin domain of the alpha(M) chain with the corresponding domain from the alpha(X) chain (p150) revealed that the activity of the alpha(M)beta(2) integrin toward chilled platelets resides within the lectin domain and does not require the I domain. Additional evidences for this conclusion are: 1) Sf9 cells expressing solely the alpha(M) lectin domain bound chilled platelets, and 2) soluble recombinant alpha(M) lectin domain inhibited the phagocytosis of chilled platelets by alpha(M)beta(2)-expressing THP-1 cells, whereas I domain substrates showed no inhibitory effect. Therefore chilled platelets are removed from blood by an interaction between beta-GlcNAc residues on clustered GPIbalpha and the lectin domain of alpha(M) chain of the alpha(M)beta(2) integrin, distinguishing this interaction from those mediated by the alpha(M) I domain.     

Mac-1 promotes FcgammaRIIA-dependent cell spreading and migration on immune complexes

The integrin Mac-1 plays a critical role in Fc receptor (FcR)-mediated antibody-dependent cellular cytotoxicity (ADCC). However, the mechanism by which Mac-1 facilitates the functions of FcgammaRIIA, a major FcR expressed on human leukocytes, is not fully understood. We report here that Mac-1 sustains cell adhesion, enhances cell spreading, and accelerates cell migration on preformed immune complexes (ICs) by directly interacting with FcgammaRIIA but not with the IC substrate. Coupling Mac-1 to FcgammaRIIA allows FcgammaRIIA to reside in the leading front of actin polymerization at the filopodial extension and thus could potentially enhance FcgammaRIIA-mediated cell spreading and migration. The direct interaction between Mac-1 and FcgammaRIIA is demonstrated by co-immunoprecipitation, by cell surface co-localization, and by solid-phase binding assays using recombinant alpha(M)I-domain and soluble FcgammaRIIA. Further mutational analysis identifies the E(253)-R(261) sequence within the alpha(M)I-domain as part of the FcgammaRIIA binding interface within Mac-1. Altogether, these results demonstrate that FcgammaRIIA recognizes Mac-1 via the alpha(M)I-domain but not the lectin domain, a distinct feature from other FcRs, and that Mac-1 binding confers FcgammaRIIA with the ability to prolong cell adhesion as well as to spread and migrate on the ICs, leading to effective cell killing by ADCC.     

Galectin-4-Regulated Delivery of Glycoproteins to the Brush Border Membrane of Enterocyte-Like Cells

We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-β- d -galactopyranoside (GalNAcα- O -bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N -glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic–recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N -glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.     

CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.     

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.     

Mac-2 binding protein is a novel e-selectin ligand expressed by breast cancer cells

Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.     

Regulation of macrophage activation markers by IL-4 and IFN-gamma is subpopulation-specific

We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.     

Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by ¹²⁵I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.     

A study of membrane glycoproteins from the rat brain using D-galactose-specific lectin

Immobilized D-galactose-specific lectin from Zea mais was used to purify rat brain membrane glycoproteins. The membrane glycoproteins preliminarily washed from soluble proteins were solubilized consecutively by 2% triton X-100 and 1% SDS. PAG-electrophoresis with SDS and 2-mercaptoethanol revealed 10 polypeptide bands (Mm 109, 62, 59, 54, 51, 42, 16, 14, 12.5 and 12 kDa) in the membrane fraction of glycoproteins solubilized with triton X-100. Additional solubilization with SDS revealed 3 bands (Mm 109, 62, and 54 kDa). Only 3 polypeptide bands (Mm 62, 59, 42 kDa) were identified when analogous procedure was used for purification of the rat liver glycoproteins. Horse radish peroxidase labelled D-galactose-specific lectin from Zea mais was found to bind to neuron bodies and neurites in the cerebellum. It is suggested that the identified brain-specific membrane glycoproteins may take part in the cell adhesion between neurons.