Specific glycoprotein changes during development of the chick neural retina

After separation of whole proteins of chick neural retina by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a number of glycoproteins can be detected by staining the gels with 125I-labeled wheat germ agglutinin (WGA) and other lectins. The glycoprotein patterns show both quantitative and qualitative changes between days 7 and 13 of development. Some of these glycoproteins can be separated by chromatography on columns of insolubilized lectins. These observations suggest that purification of some of these glycoproteins identified by staining with radioactive lectins would yield retinal antigens which may be specific for developmental stage and cell type.     

Identification of liver plasma membrane glycoproteins which bind to 125I-labelled concanavalin A following electrophoresis in sodium dodecyl sulfate.

Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Comparative analysis of the membrane proteins and their specificities in neurons,protoplasmic astrocytes,and oligodendrocytes from rat brain

Plasma membranes from neuronal perikarya (N), protoplasmic astrocytes (A) and oligodendrocyies (O) of rat brains were analysed with respect to their protein and glycoprotein contents and specificities. SDS-polyacrylamide gel electrophoresis revealed a total number of 23, 17, and 17 major proteins in N, A, and O respectively. Periodate-Schiff's staining showed that approximately 40–60% of these proteins were glycoproteins. The reactivity of these glycoproteins to Con A and WGA was also studied. Selective iodination of whole cells followed by electrophoresis and autoradiography indicated that of the major proteins, only 25% of neuronal and 60% of astroglial and oligodendroglial membrane proteins were exposed outside the cell surface. The overall results suggest that membrane proteins of each of the three cell types studied here have characteristically different internal and external markers differing in size, glycoprotein content, and reactivity of the glycoproteins to lectins.     

Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with 125I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/[3H]NaBH4. Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects.     

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.     

Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions.

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.     

Evidence for a role of the major glycoprotein in the structural maintenance of the pig zona pellucida

The functional domains of the glycoproteins of the pig zona pellucida have been analysed using lectin binding, peptide mapping, and immunoblotting in conjunction with analysis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and protein detection with the silver-based colour stain. Two of the pig zona pellucida glycoproteins identified in 2D-PAGE were differentially proteolysed within the intact matrix by a variety of enzymes. This proteolysis of specific proteins, however, did not affect the suprastructure of the matrix, or inhibit spermatozoa from adhering to the surface of the zona pellucida. The major glycoprotein appears to be involved in the structural maintenance of the zona pellucida because dissolution of the matrix correlated with proteolysis of this glycoprotein by proteinase K. These glycoproteins were further evaluated by lectin blotting with Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) before and after proteolysis of zona pellucida with trypsin. The lectins bound to all charge species of the three major zona pellucida glycoproteins. Only the most acidic components of the major glycoprotein family, which are not extensively digested, were recognized by these lectins after proteolysis. These studies provide evidence that the major glycoprotein family I of the pig zona pellucida is primarily responsible for maintaining the integrity of the matrix.     

Demonstration of feline and canine platelet glycoproteins by immuno- and lectin histochemistry

Canine and feline platelet cytocentrifuge preparations (CCPs), cryostat and paraffin-embedded bone marrow sections were used in this study. We evaluated whether platelets, megakaryocytes and megakaryocyte precursor cells could be labelled by monoclonal antibodies (Y2/51, CLB-thromb/1, HPL1) against human platelet membrane glycoprotein GP IIIa and the GP IIb/IIIa complex or by the following 10 biotinylated lectins: concanavalin A (Con A), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PsA), wheat germ agglutinin (WGA), peanut agglutinin (PNA), Phaseolus vulgaris lectin (PHA-L), Ricinus communis agglutinin 120 (RCA₁₂₀), Ulex europaeus agglutinin — I(UEA-1), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). Monoclonal antibodies Y2/51 and HPL1 cross reacted with platelets and megakaryocytic cells from both species, whereas CLB-thromb/1 was unreactive with canine preparations. Only Y2/51 labelled megakaryocytic cells in paraffin-embedded samples. LCA, PSA, WGA and PHA-L labelled feline and canine platelets and different numbers of morphologically identifiable megakaryocytes and numerous other, mostly myeloid, cells. Immunoblots of dog and cat platelet lysates using Y2/51 visualized a single protein of 95 kDa (unreduced), a mol·wt value within the range of those reported for GP IIIa. Some of the platelet (but not necessarily megakaryocyte) glycoproteins reacting with LCA, PSA and WGA could be identified in lectin blots following one- or two (nonreduced/reduced)-dimensional sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE). Thus in dogs and cats, the immunohistochemical detection of GP IIIa (and eventually GP IIb/IIIa) rather than lectin binding patterns could be important for the diagnosis of megakaryoblastic leukaemias.     

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).     

Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Analysis of Human T-Cell Antigens by One- and Two-Dimesional Polyacrylamide Gel Electrophoresis

One- and two-dimensional polyacrylamide gel electrophoresis (PAGE) was performed on immunoprecipitates formed between anti-human T-cell xenoantiserum (ATS) and cell-surface glycoproteins of human lymphocytes, that had been radioiodinated by lactoperoxidase and purified on a Lentil lectin-coupled Sepharose 4B column. In some experiments, the cells were ³H-labeled by periodate-tritiated borohydride. ATS that was absorbed with B cells recognized a number of cell-surface antigens expressed preferentially on human thymus and T cells, with molecular weights of 150K (T150), 94K (T94), 72K (T72), and 65K (T65) daltons. Whereas T150 appeared to consist of multiple components of heavily sialylated glycoproteins and to be expressed largely on thymus and T cells, and to a much lesser extent on B cells, the remaining T94, T72, and T65 glycoproteins seemed to be present on thymus and T cells but absent from B cells. Two-dimentional PAGE analysis of these T-cell glycoproteins precipitated by ATS demonstrated that T94 was an acidic glycoprotein with pI of 4, while T72 and T65, the latter being found on thymus and T cells but not on T cell-type leukemic cells, exhibited marked electric charge heterogeneity with pI ranging from 4 to 7. These data clearly suggest that human thymus and T cells possess a complex antigenic make-up on their cell surfaces, comparable to that of mouse T cells with a variety of Ly antigen systems.     

Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

Study of membrane glycoproteins of human erythrocytes using lectins

A method to study the glycoprotein composition of cell membranes, in particular of human red blood cells, has been developed. It includes the separation of membrane components by the SDS-polyacrylamide gradient slab gel electrophoresis, electroblotting of the phoretograms onto the nitrocellulose sheets and detection of glycoprotein fractions with FITC and peroxidase labeled lectins. PNA detected asialoglycoproteins with O-linked oligosaccharide chains, corresponding to all the PAS-positive bands of the phoretogram. SBA interacted more selectively and revealed only certain PAS-positive bands. Glycoproteins with N-linked carbohydrate chains were PAS-negative and can be identified only by the interaction with WGA, LCL, RCA. Group-specific agglutinins have shown that the ABO antigenic determinants are located in N-linked carbohydrate chains of membrane glycoproteins.     

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.     

FRACTIONATION OF SOLUBLE BRAIN GLYCOPROTEINS BY AFFINITY AND HYDROXYLAPATITE CHROMATOGRAPHY

—A separation of soluble brain proteins and Con A-binding glycoproteins by chromatography on calcium hydroxylapatite in the presence of SDS is described. Seventeen Coomassie Blue-stained bands were detected by polyacrylamide gel electrophoresis in SDS of Con A-binding glycoproteins of the soluble fraction of rat brain: 16 of these were found by in vivo uptake of [³H]fucose to be fucosylglycoproteins. Hydroxylapatite chromatography yielded several glycoprotein pools, each of which was shown by gel electrophoresis to contain between 4 and 8 individual glycoproteins. Such pools were enriched in [³⁶H]fucose relative to the brain soluble fraction by factors of between 6 and 21. Preliminary experiments demonstrate that this method is also applicable to the fractionation of membrane-bound glycoproteins.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

Urinary glycoproteins in acute leukemias: a 41 000 dalton glycoprotein follows the kinetic of cytoreduction

Glycoproteins of leukemic cells and 24-hour urinary proteins were subjected to SDS polyacrylamide gel electrophoresis followed by affinity labelling I125 with Concanavalin A, indicating glycoproteins with mannose and/or glucose carbohydrate residues. Among the cellular glycoproteins a 41 000 dalton glycoprotein appeared under induction therapy in close correlation to the reduction of leukemic cells in ALL as well as in AML.     

Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications

The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed.