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Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions.

The cell surface is normally covered with sugars that are bound to lipids or proteins. Surface glycoproteins play critically important roles in many cellular events, including cell-cell communications, cell-matrix interactions, and response to environmental cues. Aberrant protein glycosylation on the cell surface is often a hallmark of human diseases such as cancer and infectious diseases. Global analysis of surface glycoproteins will result in a better understanding of glycoprotein functions and the molecular mechanisms of diseases and the discovery of surface glycoproteins as biomarkers and drug targets. Here, an enzyme is exploited to tag surface glycoproteins, generating a chemical handle for their selective enrichment prior to mass spectrometric (MS) analysis. The enzymatic reaction is very efficient, and the reaction conditions are mild, which are well-suited for surface glycoprotein tagging. For biologically triplicate experiments, on average 953 N-glycosylation sites on 393 surface glycoproteins per experiment were identified in MCF7 cells. Integrating chemical and enzymatic reactions with MS-based proteomics, the current method is highly effective to globally and site-specifically analyze glycoproteins only located on the cell surface. Considering the importance of surface glycoproteins, this method is expected to have extensive applications to advance glycoscience.

Influenza is a rapidly mutating RNA virus causing mortality and morbidity in humans.The three different strains of influenza are characterized by different surface glycoproteins. Hemagglutinin (HA)...

Previous analyses of HIV-1 surface glycoprotein indicate that both the V1/V2 region and the interaction of gp120 with CD4 influence the accessibility of the V3 region on gp120. In this study we investigated the accessibility of the V3 region of HIV-2 recombinant gp125 proteins using V3-specific mAbs (7C8 and 3C4) and analyzed the binding kinetics of soluble CD4 (sCD4) to recombinant HIV-1 gp120 and HIV-2 gp125 proteins by surface plasmon resonance (SPR) analysis. Our results indicated that 7C8 recognized monomers of gp125 and gp125Delta v1v2, (lacking the V1/V2 region) while 3C4 was sensitive to the conformation of gp125, recognizing only oligomers of gp125Delta v1v2. Furthermore, SPR analysis of 7C8 binding to gp125 demonstrated that the deletion of the V1/V2 region did not increase the accessibility of the V3 region in gp125Delta v1v2. Comparative SPR analyses of sCD4 binding HIV recombinant surface glycoproteins revealed a lower affinity of sCD4 to gp125 as compared to gp120. Moreover, the analyses suggest that conformational changes only occur in HIV-1 gp120 upon interaction with CD4. We hypothesize that the V3 region is accessible in HIV-2 gp125 and thus may not require interaction with CD4 to induce conformational reorientation of the V1/V2 region.

The variant surface glycoprotein of African trypanosomes has a glycosyl phosphatidylinositol (GPI) anchor that is unusual in that its fatty acids are exclusively myristate. We showed previously that the myristate is added to a free GPI in a fatty acid remodeling reaction involving deacylation and reacylation, forming glycolipid A, the anchor precursor. We now demonstrate that trypanosomes have a second pathway for GPI anchor myristoylation distinct from the fatty acid remodeling pathway, which we call "myristate exchange." This reaction involves exchange of myristate into both the sn-1 and sn-2 positions of glycolipid A, which already contain myristate. Myristoyl-CoA, the probable myristate donor in the exchange reaction, has an apparent Km of about 6 nM. We have now identified a lyso-GPI, named theta', which has myristate as its sole fatty acid; the kinetics of formation and utilization of theta' are consistent with it being an intermediate in exchange. Myristate exchange and fatty acid remodeling appear to occur in different subcellular compartments, and the two reactions have different sensitivities to inhibitors. The myristate exchange reaction may be a proofreading system to ensure that the fatty acids on variant surface glycoproteins are exclusively myristate.

Storage of single-donor platelet concentrates is currently limited to 5 days. During this period, however, numerous morphologic and biochemical changes have been observed. These changes result in functional impairment of stored platelets. The present study describes increased binding of a monoclonal antibody against GMP 140 on the surface of stored single-donor platelets revealing an activation process. In contrast, binding of monoclonal antibodies directed against glycoprotein complex (GP) IIb-IIIa and ligand-induced binding site (LIBS1) is slightly diminished during storage. When platelets are stimulated with ADP GMP 140, GP IIb-IIIa, and LIBS1 are expressed to a higher extent than on the surface on non-stimulated platelets. The quantity exposed, however, depends upon the storage time. It is significantly reduced when platelets are stored for longer than 1-2 days. The present data indicate that storage of single-donor platelet concentrates affects fibrinogen binding, cell to cell cohesion, and release reaction. The results are in good agreement with conventional aggregation and in vitro bleeding time measurements.

The first information on surface glycoproteins came from studies of animal cell surface coats. It came from histochemistry and electron microscopy (EM). Both effective EM resolution (~20A) and selective ferritin staining for the cell surface coat promoted studies on the topic (Luft, 1976).

Identification of endogenous glycan-binding ligands for cell-surface receptors has been difficult. Incorporation of a photoactive sialic acid analog into B-cell surface glycoproteins suggests that CD22 molecules may cluster by binding carbohydrate antigens on neighboring CD22 molecules.

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).

Crystals were produced from variant surface glycoproteins (VSG) of Trypanosoma brucei brucei antigenic variants MITat 1.2, 1.6, and ILTat 1.22, 1.23, 1.24, 1.25, and 1.26. Purified VSGs had molecular weights from 60,000 to 68,000 on sodium dodecyl sulfate-polyacrylamide gels, whereas the crystals obtained were composed of polypeptides of approximate Mr 40,000-50,000. Amino-terminal amino acid sequences determined from the crystallized VSGs were identical to sequences obtained from the respective intact proteins, indicating that the crystals contained VSG amino-terminal fragments. Crystallization conditions and lattice dimensions of the crystals are given.

The influence of two kinds of transplantable melanomas on the content of fucose in the surface glycoproteins of hamster peritoneal macrophages was studied. An increase in the content of fucose, more prominent in surface material of macrophages from animals with melanotic melanoma was found. It has been suggested that this phenomenon is connected with the neoplastic process, and that the different susceptibility of the macrophages from experimental animals to macrophage inhibitory factor (MIF) is related to changes in the content of fucose in the surface glycoproteins.

Ejaculated spermatozoa of boars, bulls, rabbits, and rams were embedded in glycolmethacrylate and thin sections stained with phosphotungstic acid at low pH in order to observe the distribution of glycoproteins of the plasma membrane. Colloidal iron hydroxide was also used to detect the free acidic groups present on the sperm surface. Species-specific patterns of localizations of glycoproteins and linked negative charges were observed. The distribution was sometimes homogeneous as in bull, but generally heterogeneous in the other species. The significance of the results on sperm surface components and the practical interest to know their normal distribution are discussed.

In our isolation procedure, the surface antigens of the variants AnTat 1.1 and 1.10 ( Trypanosoma brucei brucei ) are essentially obtained as a disulfide-linked dimer while the AnTat 1.8 surface antigen is found as a mixture of monomer and disulfide-linked dimer. This observation may be related to the localization of the cysteine residues in the protein sequences. In the purification procedure using concanavalin-A Sepharose chromatography, besides the VSG elution by methyl-α- d -mannopyranoside, a quantitative elution of still bound VSG may be obtained by the addition of β-mercaptoethanol to methyl-α- d -mannopyrannoside in the elution buffer. The surface antigen of the variant AnTat 1.1 was examined for molecular form at several different times during the release procedure. The disulfide-linked dimer could be observed within 30 min of the surface coat release, indicating its presence within the parasite.

Summary The influence of two kinds of transplantable melanomas on the content of sialic acids in the surface glycoproteins of hamster peritoneal macrophages was studied, and the ability of these cells to form spontaneous rosettes. It has been found that the intensification of rosette formation by macrophages with SRBC depends on the melanoma line. No correlation has, however, been found between changes in the sialic acids content and the ability of macrophages to form rosettes under the influence of melanomas differing in malignancy.

African trypanosomes evade their mammalian host's immune system by the sequential expression of alternative surface glycoproteins. Comparison of the cDNA sequences of five antigenically distinct surface proteins and the C-terminal amino acid sequences of four additional ones demonstrates that these nine glycoproteins can be classified into two subsets based on C-terminal amino acid homologies that extend 75–105 amino acids into the protein.

Summary Variant surface glycoproteins of the African trypanosomes are membersof a multigene family which show extraordinary amino sequence diversity. The extent of this diversity and the significance of homologies both in the amino acid sequence and in the post-translational modifications are discussed in the light of what is predicted for the structure of these molecules and what is now known from X-ray crystallographic analysis.

We labeled surface glycoproteins of normal human blood platelets, granulocytes, monocytes, T and B lymphocytes, and null cells by the galactose oxidase-NaB3H4 and periodate-NaB3H4 labeling techniques. The labeled glycoproteins were observed by fluorography after separation by polyacrylamide slab gel electrophoresis. All major types of human leukocytes showed different and characteristic surface glycoprotein patterns. These patterns evidently also include common components.

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.

Variant surface glycoproteins (VSG) of Trypanosoma brucei are released in a water soluble form on impairment of membrane integrity. We have previously shown that this release is the result of an enzyme-mediated event which converts the hydrophobic membrane form VSG into the hydrophilic water-soluble form. We now present further details of the methods by which membrane form VSG ( mfVSG ) may be isolated, uncontaminated by water-soluble VSG ( sVSG ). The sensitivity to different metal ions of the enzyme that mediated the conversion event is discussed, and some biochemical characteristics of different mfVSG preparations are presented.

1. 1. Resistance to concanavalin A was accompanied by modifications to the fundamental growth and membrane-associated properties of two concanavalin A-resistant cell lines which were independently selected by either single step or cycling procedures. 2. 2. Using the galactose-oxidase [3H]borohydride surface labelling procedure, several modifications in surface glycoproteins were detected on variant cells when compared to parental wild type cells; a prominent difference was the presence of an additional surface component, with an apparent molecular weight of 155,000 on variant cells which was missing from both wild type populations. 3. 3. These observations provide strong support for the view that modified cell surface glycoproteins are directly involved in the complex concanavalin A-resistant phenotype which includes changes to some fundamental biological properties; concanavalin A-resistant variants provide an opportunity to correlate changes in membrane function with modifications in membrane structure.

The variant surface glycoproteins (VSGs) of both metacyclic and bloodstream forms of Trypanosoma congolense are shown to be anchored to the plasma membrane through a glycolipid similar to that found in Trypanosoma brucei. Release of soluble VSG from both metacyclic and bloodstream forms is associated with the exposure of an antigenic determinant homologous to the cross-reacting determinant of T. brucei VSGs. Release of soluble VSG of T. congolense can be achieved by lysates of both bloodstream and metacyclic forms of T. congolense, by lysates of T. brucei bloodstream forms, but not by lysates of procyclic forms.

The secondary structure of seven variant surface glycoproteins (VSGs) of trypanosomes has been determined by Raman spectroscopy. They are all predominantly alpha-helical, the alpha-helix content varying between 50 and 60%. The beta-strand content varies between 20 and 25%, and the content of beta-turn and nonregular structures is about 25%. For three VSGs the N-terminal domain obtained by proteolytic cleavage was found to have essentially the same secondary structure as the complete VSGs. For three VSGs a secondary structure prediction has been performed applying the rules of Chou and Fasman. In all cases, two long alpha-helices extending over about 50 residues or 80 A are predicted in agreement with the X-ray diffraction data of Freymann et al. [(1984) Nature 311, 167-169] and Metcalf et al. [(1987) Nature 325, 84-86]. The region between the two alpha-helical segments exhibits a high potential of beta-turns, suggesting that this segment may be exposed on the cell surface and carry major antigenic determinants.

Several of the recently described leucocyte surface (glyco)-proteins with significant amino acid sequence similarity (human CD9, CD37, CD53, CD63, TAPA-1, CO-029 and R2 and several homologues of other species) are distinguished by the polypeptide chain apparently four times crossing the membrane. Although the biological role of none of these molecules is known, their structure, associations with other membrane components and the effects of specific monoclonal antibodies suggest that they may constitute a family of ion channels or other transport molecules.

Sequential expression of alternative antigens enables African trypanosomes to evade the immune response. Characterisation of surface glycoproteins exposes the molecular basis of anligenic variation.

Parasitic African trypanosomes play havoc with the immune defence of their host by the consecutive synthesis of more than 100 different surface glycoproteins. Activation of the genes for some of these glycoproteins occurs through gene duplication and transposition into an expression site located elsewhere in the genome.

This study aimed to investigate high molecular weight surface glycoprotein (S-GP) patterns on various types of human leukocytes. S-GP were externally labelled by the Galactose-oxidase-NaB3H4 technique. Results based on the analysis of 120 samples derived from different types of normal and malignant leukocytes indicate that (i) the relative expression of high molecular weight S-GPs changes during haemopoietic cell differentiation and (ii) to some extent these changes enable the classification of human leukocytes.

Variants of B 16-F 1 mouse melanoma cells selected for resistance to wheat germ agglutinin (WGA) toxicity in vitro were found to have undergone a stable surface change correlated both to lectin resistance and reduced metastasizing potential. The surface alteration, as indicated by the increased electrophoretic mobilities of several lactoperoxidase-iodinated cell surface proteins in SDS-PAGE, was restricted to polypeptides able to interact with WGA. The availability of lectin-resistant melanoma cell variants having altered metastasizing behavior provides a promising approach to studies of the role of specific cell surface components in the metastasizing process.

Chinese hamster ovary cells were examined ultrastructurally following several detachment procedures. Alterations in the surface glycoproteins were observed by using ruthenium red in the fixation procedure. Trypsin removed a major portion of the cells glycocalyx and formed spherical cellular configurations. EGTA detached cells were also spherical, however, their glycocalyx appeared to remain although redistributed over the cell surface. Kinetic studies showed no alterations in subsequent population doubling times following either detachment procedure. EGTA may thus represent the current method of choice for cell detachment if preservation of the surface glycoproteins is of interest.

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.

High molecular weight (HMW) cell surface glycoproteins from 125 I-labelled normal mouse lymphocytes or cloned T helper or cytotoxic cells show different banding patterns after electrophoresis in sodium dodecyl sulphate-containing polyacrylamide gels (SDS-PAGE). Peptide mapping was used to show that these variations were due to the presence of different forms of closely-related proteins on different cell types.

The predominant molecule exposed to antibody on the surface of Trypanosoma brucei is a glycoprotein of about 60 000 molecular weight which varies in amino acid sequence. The complete sequences of two such variable surface glycoproteins (VSGs) from randomly isolated, different antigenic types of trypanosomes were compared by amino acid sequence alignment. Homologous sequences were found distributed over various regions of the VSGs. Particularly good homology was observed between residues 16-34, 91-115, 177-194 and 254-345 from the N-terminus, in addition to the known conserved region close to the C-terminus. Homology was also demonstrated in the corresponding regions of the cDNA sequences by matrix analysis.

We have begun to analyze the carbohydrates on the surface of mouse peritoneal macrophages with the long-range intent of implicating specific carbohydrates in macrophage surface function. As an initial approach to this problem, populations of resident and activated macrophages were surface labeled by the galactose oxidase and neuroaminidase/NaB3H4 method. Surface glycoproteins labeled by this method were digested with pronase and the resultant glycopeptides analyzed by gel-filtration chromatography. The results indicate that the macrophage surface is enriched in high MW lactosaminoglycan-containing glycopeptides; upon activation a marked increase in the amount of labeling and degree of branching of these lactosaminoglycans is observed.

Radioactive surface and metabolic labeling techniques were used to study the surface glycoproteins which remain in detergent-resistant cytoskeletons of cultured human fibroblasts. A 140 kilodalton (kd), fucose-containing glycoprotein (gp) was enriched in the cytoskeletal preparations together with extracellular matrix fibronectin. The 140 kd gp resisted trypsintreatment and was present as a major surface glycoprotein also in cytoskeletons of newly attached cells which had deposited only minimal quantities of extracellular matrix. In isoelectric focusing the protein was separated into three spots. The results suggest that the 140 kd gp is a cytoskeleton-associated surface glycoprotein which may play a role in the attachment of the cytoskeleton to the growth substratum.

Appearance of large Asn-linked sugar chains in the surface glycoprotein is one of the most widely found phenomena in malignant cells and is called the Warren-Glick phenomenon. Comparative study of the sugar chain structures of the surface glycoproteins of cultured cells and their malignant transformants revealed that the altered glycosylation is induced by enhancement of N-acetyl-glucosaminyltransferase V. The change seems to be highly correlated with the metastatic potential of tumor cells.

Addition of dibutyryl cyclic AMP (db-cAMP) plus theophylline to normal or virus-transformed 3T3 mouse fibroblasts, in concentrations sufficient to alter the growth characteristics, produced no qualitative changes in the pattern of incorporation of glucosamine (Glc.NH2) into glycoproteins at their extreme periphery. Nor did these drugs qualitatively alter the patterns of incorporation of 1-14C-galactose (Gal) into the principal glycosphingolipids (GSL) of these cells. In particular, virus-transformed 3T3 cells, which do not synthesize certain complex gangliosides made by normal cells, were not caused to do so. It may, however, be significant that drug treatment did result in increased incorporation of precursor galactose into complex heteroglycans of both normal and virus-transformed cells. The implications of these observations are discussed.

We have labelled the exposed surface glycoproteins of human blood T- and B-lymphocytes and cells from patients with chronic lymphocytic leukemia by the galactose oxidase-tritiated sodium borohydride method. The labelled glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography. The T- and B-lymphocytes show different and characteristic surface glycoprotein patterns. The surface glycoprotein patterns of the leukemic cells differ from those of normal, non-malignant lymphocytes. A relationship between the altered surface glycoprotein pattern of leukemic cells and the expression of leukemia-associated antigens is discussed.

Publisher Summary Lectins are defined as proteins or glycoproteins of plant (phytohemagglutinins) or animal origin displaying more or less selective affinity for a carbohydrate or a group of carbohydrates. The literature on purification of glycoproteins by use of lectins is already substantial and is growing rapidly. Because detailed procedures are usually found in the primary publications, the main intention of this article is to survey some of the approaches that have been successful. Lectins have been used extensively in studies of the properties of cell surfaces and in attempts to monitor changes in location and composition of surface glycoproteins associated with neoplastic transformation. The literature on this particular topic is burgeoning.