Lectin binding and surface glycoprotein pattern of human macrophage populations

Summary In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations.Abbreviations AM alveolar macrophage - BM blood monocyte - PM peritoneal macrophage - PBS phosphate buffered saline - IPA 12-O-tetradecanoylphorbol-13-acetate - Con A Concanavalin A - HPA Helix pomatia agglutinin - LPA Limulus polyphemus agglutinin - PHA Phaseolus vulgaris agglutinin - SBA Soy bean agglutinin - UEA I Ulex europaeus agglutinin I - WGA Wheat-germ agglutinin     

Changes in the expression of cell surface sialoglycoproteins during transition of human monocytes into macrophages

Cell surface sialoglycoproteins of human mononuclear phagocytes in different maturation stages were labelled by the periodate/borohydride method and separated by SDS-polyacrylamide gel electrophoresis. The main surface glycoproteins of peripheral blood monocytes had molecular masses of 115 and 95 kDa. During in vitro transition into adherent macrophages, the monocyte-characteristic surface glycoproteins disappeared. Most of the changes in the surface glycoprotein pattern occurred during the first 24 h and after 96 h the changes were completed. The major sialoglycoproteins of the macrophage cell surface had molecular masses of 130 and 55 kDa. The macrophage cell surface showed further changes when cultured in the presence of synovial fluid (10%). These results may reflect the in vivo maturation of monocytes into tissue macrophages. In synovium, tissue-derived factors may also take part in differentiation.     

Changes in surface antigens on preimplantation mouse embryos.

Changes in the expression of specific cell surface antigens on preimplantation mouse embryos were examined by immunocytochemistry. Embryos were recovered at various times during the preimplantation phase of normal pregnancy, and from pregnancies with experimentally induced delayed implantation, and were probed with a panel of monoclonal antibodies against murine leukocyte antigens. Antibodies directed against certain macrophage surface glycoproteins (i.e., Mac-2 and Mac-3) and those against lysosome-associated membrane glycoproteins (i.e., LAMP-1 and LAMP-2) reacted specifically with cell surface determinants on the embryos. Differences in the spatiotemporal patterns of antibody binding during normal and delayed implantation indicate that expression of the antigenic determinants recognized by these antibodies is regulated individually in response to intrinsic as well as extrinsic signals at the time of implantation, and thus they may be important for the process of embryo implantation.     

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.     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

A new method for quantitative analysis of cell surface glycoproteome

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface‐capturing strategy where the glycopeptides were submitted to LC‐MS/MS analysis directly for identification of glycoproteins and the non‐glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.     

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.     

Analysis of cell surface glycoprotein changes related to hematopoietic differentiation

A high-resolution technique has been used to study differentiation-related and leukemia-associated glycoproteins. Cells are labeled with the membrane-impermeable probe sulfo-N-hydroxysuccinimidyl-biotin. Nonionic detergent extracts are subjected to affinity chromatography on a number of immobilized lectins and after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) and western transfer, the biotin-labeled glycoproteins are visualized by using avidin-horseradish peroxidase and 4-chloronaphthol. With the aid of the lectins concanavalin A, Dolichos biflouros agglutinin, Lens culinaris hemagglutinin, peanut agglutinin, pokeweed mitogen, Ricinus communus agglutinin I, soybean agglutinin, Ulex europeus agglutinin I (UEA), and wheat germ agglutinin, each purifies different glycoprotein subsets from the same cell type. Mature cells of distinct hematopoietic lineages differ considerably in their cell surface glycoprotein patterns. This technique was used to analyze the glycoproteins of human leukemia cells before and after the induction of differentiation. K562 cells differentiated along different lineages after treatment with phorbol 12-myristate 13-acetate, sodium butyrate, dimethyl sulfoxide, or hemin. Limited specific alterations were observed with a number of lectins when K562 erythroleukemia cells were induced to differentiate. Among these, a number of bands were identified that were either lost or appeared after induction of differentiation with all four agents. In contrast, the glycoproteins bound by UEA were drastically diminished after induction of differentiation, and the remaining UEA-bound glycoproteins bore little resemblance to those of the cells before treatment. This high-resolution technique may be useful as a general method for the examination of cell surface glycoprotein differences. Once specific glycoprotein alterations are detected, lectin affinity chromatography and SDS-PAGE allow purification of antigens for the production of monoclonal antibodies.     

The third chains of living organisms--a trail of glycobiology that started from the third floor of building 4 in NIH

Application of a finger-printing method to the analysis of human milk oligosaccharides led to the finding that several oligosaccharides were missing in the milk of non-secretor or Lewis-negative individuals. This finding helped us in opening the door of elucidating the enzymatic basis of blood types in human. Based on these successful studies, a strategy to establish reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins was devised. It was to contrive enzymatic and chemical means to release quantitatively the N-linked sugar chains as oligosaccharides, and finger-print them by using appropriate methods to demonstrate the sugar pattern of a glycoprotein. These methods enabled us to determine that the N-linked sugar chains of glycoproteins can be classified into three subgroups: high mannose-type, complex-type, and hybrid-type. By comparative studies of the sugar patterns of a glycoprotein produced by different organs and different animals, occurrences of organ- and species-specific glycosylation were found in many glycoproteins. By comparative studies of the glycosylation patterns of the subunits constructing human chorionic gonadotropin and other glycoproteins, occurrence of site-directed N-glycosylation was also found, indicating that the processing and maturation of the N-linked sugar chains of a glycoprotein might be controlled by the structure of polypeptide moiety. Furthermore, these methods enabled us to elucidate the structural alteration of the sugar chains of a glycoprotein induced by diseased state of the producing cells, such as rheumatoid arthritis and malignancy. Recent studies of glycoproteins in the brain-nervous system through aging revealed that N-glycosylation of P(0) in the rat spinal cord is induced by aging. Therefore, glycobiology is expanding tremendously into fields such as pathological and gerontological research.     

Schistosoma mansoni surface glycoproteins. Analysis of their expression and antigenicity

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin-affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose-binding protein, only in the concanavalin-A-bound fractions were glycoproteins identified. Changes in concanavalin-A-binding glycoproteins were detected after transformation and early maturation of the schistosomula. Some glycoproteins disappeared (Mr 38 000, 29 000 and 25 000), some appeared independently of host molecules (Mr 19 000), others only appeared after culture in human serum (Mr 45 000). Two major glycoproteins of Mr 32 000 and 16 000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3-h schistosomula only the strong Mr-38 000-32 000 complex was found to be antigenic. Thus many major low-molecular-mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr-38 000-32 000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Carbohydrate binding specificity of the recombinant chitin-binding domain of human macrophage chitinase

The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.     

Influenza virus glycoprotein-reactive human monoclonal antibodies

Influenza continues to be a significant public health challenge. Two glycoproteins on the surface of influenza virus, hemagglutinin and neuraminidase, play a prominent role in the process of influenza virus infection and release. Monoclonal antibodies targeting glycoproteins can effectively prevent the spread of the virus. In this review, we summarized currently reported human monoclonal antibodies targeting glycoproteins of influenza A and B viruses.     

Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice

The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.     

Envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions.

Infection by human immunodeficiency virus type 1 (HIV-1) leads to progressive destruction of the CD4+ T-cell subset, resulting in immune deficiency and AIDS. The specific binding of the viral external envelope glycoprotein of HIV-1, gp120, to the CD4 molecules initiates viral entry. In the past few years, several studies have indicated that the interaction of HIV-1 envelope glycoprotein with cells and molecules of the immune system leads to pleiotropic biological effects on immune functions, which include effects on differentiation of CD34+ lymphoid progenitor cells and thymocytes, aberrant activation and cytokine secretion patterns of mature T cells, induction of apoptosis, B-cell hyperactivity, inhibition of T-cell dependent B-cell differentiation, modulation of macrophage functions, interactions with components of complement, and effects on neuronal cells. The amino acid sequence homologies of the envelope glycoproteins with several cellular proteins have suggested that molecular mimicry may play a role in the pathogenesis of the disease. This review summarizes work done by several investigators demonstrating the profound biological effects of envelope glycoproteins of HIV-1 on immune system cells. Extensive studies have also been done on interactions of the viral envelope proteins with components of the immune system which may be important for eliciting a "protective immune response." Understanding the influences of HIV-1 envelope glycoproteins on the immune system may provide valuable insights into HIV-1 disease pathogenesis and carries implications for the trials of HIV-1 envelope protein vaccines and immunotherapeutics.     

Cytochemical and ultrastructural studies concerning the cell coat glycoproteins in normal and transformed human blood lymphocytes: I. Variations of sialic acid containing glycoproteins subsequent to transformation of T and B lymphocytes by various kinds of stimulating agents

This is an ultrastructural study on variations occurring subsequent to blast transformation in the amount and/or topographical distribution of sialic acid end groups carried by the cell coat glycoproteins of human blood T and B lymphocytes as revealed by PTA staining according to the Rambourg technique.The transformation was immediately followed by a transient decrease of the surface labelling in both T- and B-derived lymph-cell lines, irrespective of the nature of the stimulating agents used for induction of transformation.Nevertheless, upon reaching their final cyto-differentiation step, cells of both lines seemed to recover their initial PTA staining patterns observed in the controls.This transient decrease in the relative number of sialic acid end groups during a period of dramatically rapid cell growth and multiplication may most plausibly be interpreted as reflecting an ‘incomplete maturation’ of the newly synthesized cell coat glycoproteins.     

Isolation and charaterization of surface glycoproteins from L-1210, P-388 and HeLa cells

A method is described that permits the rapid extration of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells.Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with ¹²⁵I by the lactoperoxidase iodination technique; (b) with ³H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells wee shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Surface glycoproteins of resting and activated human T lymphocytes

Summary This review summarizes some recent studies on the surface glycoproteins of human thymocytes and T lymphocytes. Purified cells were surface labeled by the galactose oxidase-NaB³H₄ or periodate-NaB³H₄ techniques. The radioactive membrane glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Thymocytes and T lymphocytes show characteristic surface glycoprotein profiles which are easily distinguishable from those of the other main groups of human leukocytes. We observed specific changes in the surface glycoprotein patterns which correlate with the degree of maturation and functional activation of T cells. Surface molecules carrying T cell specific antigens were identified by immune-precipitation from lysates of surface labeled thymocytes and T lymphocytes using rabbit anti-human T cell antibodies. Finally we describe a leukocyte membrane glycoprotein which is a precursor of serum ₁ acid glycoprotein (orosomucoid).     

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.