Developmentally regulated expression of a 78 kDa erythroblast membrane glycoprotein immunologically related to the platelet thrombospondin receptor.

We have previously described a monoclonal antibody (FA6-152), obtained by immunizing mice with fetal human erythrocytes [Edelman, Vinci, Villeval, Vainchenker, Henri, Miglierina, Rouger, Reviron, Breton-Gorius, Sureau & Edelman (1986) Blood 67, 56-63]. The antibody labelled fetal, but not adult, erythrocytes and bound to both fetal and adult platelets and monocytes. In the present study we have characterized the antigen recognized by FA6-152 on human platelets and on cells of the erythroid lineage at different stages of maturation. FA6-152 precipitated a chymotrypsin-resistant 88 kDa sialoglycoprotein from both iodinated and periodate/NaB3H4-surface-labelled platelets which corresponds to glycoprotein IV, the platelet thrombospondin (TSP) receptor. After neuraminidase treatment, a shift of the apparent molecular mass from 88 kDa to 85 kDa was observed. Scatchard analysis revealed that 125I-FA6-152 bound saturably with high affinity to a single class of platelet binding sites (Kd 6.4 +/- 0.6 nM). The number of FA6-152 IgG molecules bound per platelet was 25,400 +/- 8,800 (n = 4) and did not change upon thrombin activation of platelets. At low doses of alpha-thrombin (0.025 unit), FA6-152 inhibited platelet aggregation as well as endogenous TSP binding to the platelet surface. Immunofluorescence labelling of bone-marrow cells and of cultures in vitro of burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) revealed that that FA6-152 antigen is a very early marker of erythroid differentiation and that its expression declines during maturation. Immunochemical identification of the FA6-152 antigen on fetal erythroblasts and fetal mature erythrocytes revealed a 78 kDa glycoprotein migrating just in front of the glycophorin A dimer. The antigen, which was absent from adult mature erythrocytes, was also detected in human erythroleukaemic (HEL) cells where FA6-152 precipitated two bands of molecular mass 85 and 88 kDa. Our data establish the existence of a previously unidentified 78 kDa erythroblast cell-surface glycoprotein whose expression is developmentally regulated during erythroid differentiation and which is immunologically related to the 88 kDa platelet TSP receptor.     

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment

The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.     

Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study

Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19-OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib.     

Histochemical demonstration of sugar residues by lectin and immunocytochemical techniques for blood group antigens in human colon

Summary Goblet cell mucin in 39 human colons was studied by methods specific for various sugar residues, including staining with three lectins,Dolichos biflorus agglutinin (DBA, specific for blood group A antigen),Griffonia simplicifolia agglutinin-I (GSA-I, B) and peanut agglutinin (PNA, T antigen), and immunostaining for A, B, H and T. Isoantigens A, B or H were found only in the right colon. GSA-I reactive goblet cells occurred in the right colon of both blood group A and B patients and possibly contained isoantigens. However DBA reactive cells were found in all cases. Prior neuraminidase digestion imparted anti-A, GSA-I and DBA reactivities to the cells lining the lower crypts in all cases. This pretreatment also imparted PNA and anti-T reactivities to goblet cells, only the latter reactivity being eliminated by galactose oxidase. Goblet cell mucin in transitional mucosa revealed decreased A and B, and increased H antigens. Enhanced galactose oxidase—Schiff (GOS) and anti-T reactivities were also noted. The present results revealed that some lectin reactions of goblet cells might be related to blood group antigens but others were not, and that different techniques for demonstrating reputedly the same sugar residues produced different results, indicating a need for proper evaluation of their specificity.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

AN ULTRASTRUCTURAL STUDY OF EARLY MORPHOGENETIC EVENTS DURING THE ESTABLISHMENT OF FETAL HEPATIC ERYTHROPOIESIS

Morphogenetic events are described which characterize early stages of the interaction between mesenchyme and expanding epithelial cell cords derived from the hepatic endodermal diverticulum in the C57BL/6J mouse. This interaction culminates in the differentiation of hepatic epithelial and hematopoietic tissues. No basement membrane separates the presumptive hepatic epithelial cells from the adjacent mesenchyme, while intercellular attachments, both adherent junctions and desmosomes, are established transiently between heterologous cell types across this epithelio-mesenchymal interface. Yolk sac-derived erythroblasts found in the primitive liver are distinguished morphologically from endogenous hepatic erythroid cells; they are confined to the vascular compartment and are not, apparently, precursors for hepatic erythropoiesis. The earliest recognizable endogenous hepatic hematopoietic cells appear, extravascularly, among those mesenchymal cells in intimate contact with the endodermal epithelium between the 10¼ and 10½ gestational day. Definitive erythropoiesis commences between the 10½ and 11th fetal days. The ultrastructure of these primitive hepatic erythroid cells (proerythroblasts) and their transition to more mature forms (basophilic and polychromatophilic erythroblasts) are described.     

Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

 To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells. Accepted: 22 January 1997     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Differences in lectin binding in squamous metaplasia induced by benzo(a)pyrene and vitamin A deficiency in hamster tracheal explants

Summary Epidermoid metaplasia in the hamster trachea can be produced by treatment with benzo(a)pyrene (BP) or vitamin A deficiency. To elucidate distinguishing features of the two types of lesions, lectin binding to tissue sections of tracheal explants exhibiting metaplastic lesions was assessed. In squamous metaplasia induced by vitamin A deficiency, Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) showed faint (+) to moderate (++) binding in both basal and suprabasal cells; Concanavalin A (Con A) showed moderate binding (++) to suprabasal cells and no binding in basal cells. In the BP-induced lesions, PNA and WGA bound intensely (++++, +++, respectively) in basal cells and faintly (+) to moderately (++) in suprabasal cells. The staining seemed to be predominant at the periphery of the cells. Further, the intensity of PNA and WGA staining increased significantly after the neuraminidase treatment. DBA and Con A showed faint (+) to moderate (++) binding in the BP-induced metaplasia. The results show that in BP-induced metaplasia, cells in the basal region show preferential binding of PNA and WGA. This research was supported by grant RO1-HL32308 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Galactosyl-binding lectins: Potentiation of mitogenicity by erythrocyte membrane fragments

The galactosyl-binding lectins, soybean agglutinin (SBA) and peanut agglutinin (PNA), exhibit a low mitogenic activity for human peripheral lymphocytes isolated from heparinized blood. We report here that responses of lymphocytes isolated from blood defibrinated by swirling with glass beads, are enhanced up to 100-fold when stimulated with these lectins. Brief incubation of lymphocytes with defibrinated serum also results in a marked potentiation of their responses to SBA and PNA. This augmentation can be mimicked by subjecting purified lymphocytes mixed with washed human erythrocytes to the mechanical process used in defibrination. Mechanical agitation of whole blood or washed erythrocytes results in partial lysis of red blood cells, and brief incubation of lymphocytes with erythrocyte lystates also enhances responses to galactosyl-directed lectins. Sialic acid release and mitogen binding are not markedly altered in cells separated by defibrination or in those treated with erythrocyte lysates. Direct addition of erythrocyte lysates to cell cultures enhances responses to SBA but not to PNA. When neuraminidase is also added to these cultures, responses to both SBA and PNA are markedly enhanced. Our findings suggest that SBA and PNA are rendered supermitogenic by interacting with a particulate fraction that is formed by mechanical shearing of erythrocytes. These findings indicate the importance of the mode of presentation of mitogens to cells in eliciting a blastogenic response.     

Effects of alpha-L-fucosidase digestion on lectin staining in human pancreas

We examined the effects of alpha-L-fucosidase digestion on lectin staining in formalin-fixed, paraffin-embedded human pancreatic tissue from individuals of different blood groups. Digestion with the enzyme resulted in apparent diminished intensity of Ulex europaeus agglutinin-I (UEA-I) staining in the acinar cells. In addition to the decreased intensity of UEA-I staining, reactivity with soybean agglutinin (SBA) was increased in the enzyme-susceptible, UEA-I-reactive cells. The intensity of Griffonia simplicifolia agglutinin-II (GSA-II) staining performed after beta-galactosidase digestion in UEA-I-reactive acinar cells was markedly increased by prior treatment with fucosidase. GSA-II staining following sequential digestion with fucosidase and galactosidase was completely abolished by subsequent digestion with beta-N-acetylhexosaminidase. These results therefore substantiate the previous assumption that SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and GSA-II reactive beta-N-acetyl-D-glucosamine imparted following galactosidase digestion represent precursors of H antigen. The present study further demonstrated that intense peanut agglutinin (PNA) staining was imparted after digestion with fucosidase in UEA-I-reactive sites in secretors. In contrast, nonsecretors showed vivid PNA staining that was usually detected throughout the pancreas without prior enzyme digestion. Here, fucosidase digestion had if any little effect on PNA staining. These results suggest that in secretors a terminal trisaccharide, fucosylated D-galactose-(beta 1-3)-N-acetyl-D-galactosamine exhibiting positive PNA reaction after fucosidase digestion, exists in UEA-I-reactive acinar cells. It is assumed that the secretor gene could control the step of final fucosylation of D-galactose-(beta 1-3)-N-acetyl-D-galactosamine in human pancreas.     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation.