The Biochemical characterization of syrian hamster cell-surface alloantigen

The first alloantiserum to be described in Syrian hamsters has been characterized for its ability to react with externally and internally radiolabeled antigens derived from normal hamster lymphoid cells. Utilizing conventional biochemical techniques, radioiodinated and³H-leucine labeled cellular extracts have been prepared, partially purified by lentil lectin affinity chromatography, and indirectly immunoprecipitated with experimental alloantisera. Analysis of the precipitated radiolabeled antigens by polyacrylamide gel electrophoresis in SDS has identified two prominent cell-surface proteins at 39,000 (p39) and 29,000 daltons (p29) on 2-mercaptoethanol reduced gels. Further analysis of the radiolabeled extract has demonstrated the existence of hamster cell-surface proteins at 43,000 and 12,000 daltons which are immunoprecipitated by a xenoantiserum directed against human ₂ microglobulin. Coelectrophoretic studies indicate the independent identity of these four species of hamster cellsurface proteins. These results suggest that between two hamster lines, derived from animals caught 40 years apart from different geographic locations in Syria, polymorphism of cell-surface antigens is restricted to p39 and p29 molecular species.     

Alterations of cell-surface carbohydrates during differentiation and development

Expression of many cell-surface carbohydrates is controlled temporally and spatially by developmental programs. This subject is reviewed from 5 viewpoints: structural changes revealed by chemical analysis, cell-surface markers useful for cell identification and separation, core proteins carrying the developmentally regulated carbohydrate chain, glycosyltransferases responsible for the change and the biological meaning of the phenomenon. The differentiation systems covered are mainly early mammalian embryogenesis and the differentiation of blood and nerve cells.     

Site-specific distribution of epithelial cell-surface carbohydrates in rat oral mucosa

The binding of two fluorescein-labelled lectins to epithelial cell surfaces was examined microspectrofluorimetrically in rat oral mucosa. Griffonia simplicifolia (GS-I-B4), which is specific for alpha-D-galactosyl end groups, labelled only basal cells, while Ulex europaeus (UEA-I), which is specific for alpha-L-fucosyl groups, labelled only spinous cells. The degree of binding of the lectins was dependent on the lectin concentration and the lectin pH. Different sites were examined. The labelling of basal cells by GS-I-B4 was maximal on the lateral borders of the tongue, and the fluorescence diminished medially; in contrast, the UEA-I labelling of the corresponding spinous cells was of undiminished intensity in the mediolateral direction across the entire lingual epithelium. There was a gradual increase in the binding of GS-I-B4 and UEA-I towards the posterior aspect of the tongue. In the mid-palate, there was stronger staining both of basal cells by GS-I-B4 and of spinous cells by UEA-I in the gingivae as compared to the centre of the palate. In anteroposterior sections of the fore- and mid-palate, the fluorescence intensity of basal cells was inversely related to that of spinous cells, with maximal labelling of basal cells by GS-I-B4 and corresponding minimal binding of spinous cells by UEA-I being evident at the crests of the transverse rugae, and the opposite pattern of staining by both lectins being noted at the bases of the rugae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-bound carbohydrates on cell-surface as targets of recognition: An Odyssey in understanding them

Multidisciplinary approaches by a number of investigators have established that cell-surface carbohydrates are integral components of recognition systems regulating survival, migration, adhesion, growth and differentiation of various cells. Our own experience and contributions to this exciting field are described. We discovered Endo D as the first endoglycosidase acting on glycoproteins, found complementary specificity of two endoglycosidases (Endo D and Endo H), and applied these enzymes for glycoprotein research. Endo--galactosidase C, which hydrolyzes Gal1-3Gal1-4GlcNAc xenoantigenic determinant, was later found and molecularly cloned. We also found highly branched poly-N[emsp4 ]-acetyllactosamines in early embryonic cells, and demonstrated developmentally regulated carbohydrate changes during early mammalian development. The binding site for Dolichos biflorus agglutinin was introduced as a new differentiation marker. Basigin and embigin, two related members of the immunoglobulin superfamily, a sialomucin MGC-24 and other glycoproteins were discovered as carriers of developmentally regulated carbohydrate markers. We proposed enhancement of integrin action as a function of sugar chains with Lewis X epitope, and observed a relationship between the expression of carbohydrate markers and invasive properties of human carcinoma. Midkine, a heparin-binding growth factor, was discovered more recently and its interaction with heparin and oversulfated chondroitin sulfate was elucidated. N[emsp4 ]-Acetylglucosamine-6-sulfotransferase was cloned and used to reconstitute l-selectin ligands. Gene knockout was applied to reveal in vivo function of basigin, syndecan-4 and chondroitin 6-sulfate. Throughout my research on all these subjects, I have been fortunate in obtaining unexpected observations and enjoying fruitful collaborations.     

Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa

The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.     

The immunotherapeutic value of a L1210 tumor cell vaccine depends upon the expression of cell-surface carbohydrates

Summary Active, specific immunotherapy of L1210 leukemia (following reduction in tumor burden by chemotherapy) with an L1210 tumor cell subpopulation (con A-Fraction A) depended upon the integrity of cell-surface constituents. The immunotherapeutic value of con A-Fraction A cells was abrogated by treatment of the cells with either neuraminidase or galactosidase, and mice treated with these cells died faster than mice treated with chemotherapy alone. Glucosidase and protease treatment totally destroyed the immunogenicity of the tumor cell vaccine. The results suggest that the immunogenic properties of this vaccine may reside in the cell-surface expression of glycoprotein.Supported, in part, by a research contract from the National Cancer Institute, USA     

The role of carbohydrates in sperm-egg interaction in rats

The first step in fertilization is the interaction of the capacitated sperm with the zona pellucida. It has been proposed that the initial interaction, as in other types of cell adherence, is due to complementary interacting sites on the opposing surfaces of the gametes. This work intended to investigate the role of carbohydrates in sperm-egg binding in rats. Ejaculated sperm was collected from uterine horns of mated females. The sperm was suspended in Rat Fertilization Medium at final concentrations of 3-7 times 10(5) sperm/ml. After 5 1/2 h of sperm incubation, eggs were added to the sperm suspensions concomitantly with various carbohydrates to achieve a final concentration of up to 50 mM. The eggs were separated after 30 min, and the number of sperm bound to the zona pellucida was counted. Among a variety of monosaccharides tested at 50 mM concentration, it was found that alpha-methyl-mannoside was the most potent inhibitor (producing 80% inhibition); less potent was D-mannose and even less, L-fucose. A combination of alpha-methyl-mannoside and L-fucose showed a synergistic effect. Mannan was not more effective as an inhibitor than the monosugar mannose, while fucoidin was extremely potent, causing over 90% inhibition of binding at 0.1%. We assume the presence of macromolecules containing sugars on the zona pellucida because inhibition of sperm binding to this layer was observed: a) after preincubation of mannan or fucoidin with sperm, but not with the eggs; and b) after pretreatment of the egg with specific enzymes. The results obtained in this study in the rat are consistent with the hypothesis that carbohydrates are critical for the sperm-egg interaction.     

Detection of anomalies in cell-surface carbohydrates on thrombasthenic platelets using 125I-labeled lectins

The composition of carbohydrates on the surface of platelets from a patient with Glanzmann's thrombasthenia and from seven normal donors were determined and compared. To this end, binding studies were performed using nine different purified 125I-labeled lectins; Concanavalin A, P-Phytohaemagglutinin, Wheat Germ Agglutinin, Dolichos biflorus, Pisum sativum, Ricinus communis II Agglutinin, Tetragonolobus purpureus, Lens culinaris and Soybean Agglutinin. These studies show that thrombasthenic platelets bear significantly decreased numbers of receptors for Concanavalin A and Lens culinaris, both with a specificity for D-mannose, and Ricinus communis II, with specificity for D-galactose. There were no detectable differences in the numbers of other lectin receptors. These results provide further evidence of molecular defects in thrombasthenic platelets. Moreover, the use of 125I-labeled lectins, as shown here, provides a fast and reliable technique for identifying abnormalities in the carbohydrate composition on the surface of platelets in various thrombopathies.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Alterations in cell-surface carbohydrates of rat large granular lymphocytes associated with interleukin-2 activation

The activation of large granular lymphocytes (LGLs)/natural killer (NK) cells with interleukin-2 (IL-2) has been shown to increase the ability of these cells to lyse NK-resistant tumor target cells. Activated LGLs, termed LAK (lymphokine-activated killer) cells, have been demonstrated to be of therapeutic value in vivo against metastatic tumors. The mechanism by which IL-2 induces broadened cytolytic capability, as well as the molecular basis of target recognition and killing by the activated cells has not yet been elucidated. Since carbohydrate moieties have been demonstrated to be of possible significance in the cytolytic cascade of a variety of effector cells, the current study was undertaken to determine if the activation of LGLs with IL-2 is accompanied by an alteration of cell-surface carbohydrates. Two-color flow cytometry was performed to identify LGL/NK cells in populations of nylon wool-nonadherent splenic mononuclear cells and to assess the binding of various lectins to activated as well as nonactivated LGLs. Increases were observed in the binding of four lectins to LGLs after IL-2 activation; Triticum vulgaris (wheat germ agglutinin), Phytolacca americana (pokeweed mitogen), Lycopersicon esculentum (tomato lectin), and Griffonia simplicifolia I-B4 (GSI-B4). The wheat germ, pokeweed, and tomato lectins recognize complex carbohydrates structure consisting of GlcNAc(Bl,4GlcNAc)n while GSI-B4 recognizes alpha-D-galactose terminal end groups. Lectin binding to the activated LGLs was homogenous (i.e., flow cytometry revealed only a single population of fluorescent cells). Lectin binding to LGLs prior to activation was more heterogeneous, however, the tomato lectin uniquely revealed a bimodal distribution of receptors. These data indicate that LGL/NK cells from the rat are heterogeneous in their ability to bind specific lectins, and that IL-2 activation of these cells results in altered expression of specific cell-surface carbohydrates.     

Spermatogenesis in the golden hamster: the role of c-kit.

c-kit is related to the family of transmembrane tyrosine kinase receptors. Mutations in genes for either c-kit or its ligand, Steel factor, result in infertility, but the role of c-kit/SCF system in spermatogenesis is not well understood. In this study Western blot analysis together with confocal microscopy were used to follow c-kit expression in hamsters during the first spermatogenic wave in mature animals and in old age. Three antibodies raised against different domains of c-kit were tested on Western Blot. Confocal microscopy was performed after incubation of fixed seminiferous tubules with tested antibodies followed by binding of FITC-labeled secondary antibody. Longitudinal sections of seminiferous tubule were observed by confocal microscopy to determine in which stages of spermatogenesis and in which cell types c-kit was found. C-kit bands of 80,140, and 150 kDa were observed on Western blot, indicating that c-kit is a name related to several proteins sharing some common domains. Only the band of 150 kDa correlated with positive staining of c-kit in tubules using confocal microscopy. We term this protein c-kit150T (150 kDa, testis). We demonstrated that c-kit150T appeared in differentiating hamster spermatogonia at stages VII-VIII of adult spermatogenesis and at day 13-14 during the first spermatogenic wave. It remained attached to the cell until late pachytene. This suggests that c-kit may play a role in preparing the germinal cells to enter meiosis. In order to evaluate the effect of aging on the number of germ cells, B2 spermatogonia/Sertoli cell ratio was calculated in the group of young animals (5-7 months) compared to this ratio in older ones (20-26 months). A significant decrease (P < 0.01) in the number of B2 spermatogonia in the group of old hamsters as compared to young ones was seen. The calculated value for the B2 spermatogonia/Sertoli cell ratio was 5.6 +/- 0.7 in young animals and 3.8 +/- 1.2 in the 20-26 months ones. In addition, decrease in the intensity of staining for c-kit was detected in the old hamsters. These may be the reasons for subfertility in old age and in other cases of testicular disorders.     

Selection and syntheses of tentacle type peptides as 'artificial' lectins against various cell-surface carbohydrates

Sialyl Lewis X and its derivatives are cell-surface carbohydrates that are involved in cell-cell recognition by carbohydrate-mediated interactions. Unfortunately, owing to the similarities between carbohydrates only a limited number of tools are available for their differentiation. In this study, we prepared a selected phage-displayed peptide library against LeX (2), SLN (3), or LN (4), which compared to sLeX (1) lack sialic acid, fucose, and both sialic acid and fucose from constituents, respectively. Sequences of the selected peptides, prepared as tentacle type dimeric peptides, were prepared and shown to have micromolar affinities for the cognate carbohydrates. The specificities displayed by these 'artificial' lectins overwhelm those of natural lectins. These results suggest that they can serve as useful tools to detect changes in the terminal monosaccharide of cell-surface carbohydrates.     

Genetic studies of the Syrian hamster

A new mutant allele of the Syrian hamster is described. It is inherited as an autosomal recessive and is probably an homologue of the gene for brown pigment, a mutational step which is known to occur in a number of rodent species. In animals homozygous for the mutant allele, all the normal black eumelanin is changed to brown. The new coat colour engendered in this manner is described in detail. The brown allele has been tested for linkage against the genes cream and ruby-eye but the results were negative.     

Genetic studies of the Syrian hamster

A new mutant gene,anophthalmic white, is described for the Syrian hamster. The gene is inherited as a dominant to normal and, when homozygous, produces a characteristic syndrome of achromia and anophthalmia or microphthalmia. The heterozygote possesses white belly fur (instead of cream), a fine sprinkling of white hairs in the adult coat and a diminution of eye pigmentation. An occasional heterozygote may possess a small patch of unpigmented fur on the head or body. The new mutant does not appear to be linked with the gene forcream coat colour nor that forpiebald spotting. The significance of homologous mutants, with the above syndrome, in the mouse and hamster is briefly discussed.