Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.     

Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.     

Identification of surface components of mammalian respiratory tract cilia

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X-100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 51 kd. Other membrane + matrix proteins at 126 and 76 kd bound streptavidin even from nonlabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X-100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.     

Cytochemical analysis of oligosaccharide processing in frog photoreceptors

Summary Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections ofXenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with theN-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

Modulation of endothelial cell surface glycoconjugate expression by organ-derived biomatrices

Cell surface molecules play an important role in cellular communication, migration, and adherence. Here, we show the effect of organ-derived biomatrices on endothelial cell surface glycosylation. Five different lectins (with and without neuraminidase treatment) have been used as probes in an enzyme-linked lectin assay to quantitatively detect glycoconjugates on endothelial cells (BAEC) grown on tissue culture plastic or biomatrices isolated from bovine lung, liver, and kidney. BAEC generally exhibit strong binding of concanavalin A (Con A), Ricinus communis agglutinin I (RCA-I), wheat germ agglutinin (WGA), and soybean agglutinin, and peanut agglutinin after neuraminidase pretreatment of cells (Neu-SBA and Neu-PNA), while SBA and PNA consistently bind weakly to BAEC. BAEC grown on organ-derived biomatrices exhibit significantly altered binding intensities of Con A, RCA-I, WGA, and Neu-PNA: BAEC cultured on lung- or kidney-derived biomatrices express significantly stronger binding affinities for Con A and RCA-I than BAEC grown on liver-derived biomatrix or tissue culture plastic. In contrast, BAEC binding of WGA and PNA (after treatment of cells with neuraminidase) is significantly reduced when BAEC are grown on liver- or kidney-derived biomatrix. Quantitative lectin immunogold electron microscopy reveals consistently stronger lectin binding over nuclear regions compared to junctional regions between neighboring cells. These results indicate that extracellular matrix components regulate endothelial cell surface glycoconjugate expression, which determines cellular functions, e.g., preferential adhesion of lymphocytes or metastatic tumor cells.     

Differential expression of sialic acid and N-acetylgalactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential.

In this study we investigated the levels of expression of sialic acid and N-acetylgalactosamine residues on the cell surface of a normal intestinal epithelium cell line, IEC-6, and in two colon adenocarcinoma cell lines with different metastatic potential, Caco-2 and HCT-116. Glycoprotein expression was estimated initially by cytochemistry with WGA and HPA lectins and biochemistry with isolated plasma membrane fractions of the cells. Fluorescence and electron microscopic analyses revealed differences in the expression profile of carbohydrates recognized by the lectins used on the cell surface of IEC-6, Caco-2, and HCT-116 cells. Lectin blotting identified a range of eight HPA-binding glycoprotein bands with molecular weights of 16-66 kD in Caco-2 cells, six glycoproteins of 16-36 kD, and three protein bands of 35, 24, and 21 kD in IEC-6 cells. A minor band of 66 kD and a major one of 50 kD for WGA-binding glycoproteins were observed in IEC-6 cells and seven glycoproteins of 18-97 kD in Caco-2 and HCT-116 cells but with a visible higher expression of these glycoproteins in the latter. Furthermore, significant quantitative difference in levels of expression of WGA- but not of HPA-binding glycoconjugates was noted, as analyzed by high-resolution scanning electron microscopy using backscattered electron images of cells incubated with gold-labeled lectins.     

Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells

Two Chinese hamster ovary (CHO) cell mutants selected for resistance to wheat germ agglutinin (WGA) have been shown to exhibit defective sialylation of membrane glycoproteins and a membrane glycolipid, GM3. The mutants (termed WgaRII and WgaRIII) have been previously shown to belong to different genetic complementation groups and to exhibit different WGA-binding abilities. These mutants and a WGA-resistant CHO cell mutant termed WgaRI (which also possesses a surface sialylation defect arising from a deficient N-acetylglucosaminyltransferase activity), have enabled us to investigate the role of sialic acid in WGA binding at the cell surface. Scatchard plots of the binding of 125I- WGA (1 ng/ml to 1 mg/ml) to parental and WgaR CHO cells before and after a brief treatment with neuraminidase provide evidence for several different groups of sialic acid residues at the CHO cell surface which may be distinquished by their differential involvement in WGA binding to CHO cells.     

Regionalization of transmembrane glycoproteins in the plasma membrane of boar sperm head is revealed by fracture-label

We used fracture-label and surface labeling techniques to characterize the distribution and topology of wheat germ agglutinin (WGA) receptors in the plasma membrane of boar sperm heads. We show that freeze- fracture results in preferential, but not exclusive, partition of WGA- binding sites with the outer (exoplasmic) half of the plasma membrane. Labeling of the inner (protoplasmic) half of the membrane is significant, and is denser over the areas that overlie the acrosome. Exoplasmic membrane halves are uniformly labeled. Analysis of freeze- fracture replicas revealed that the distribution of intramembrane particles over protoplasmic faces parallels that of WGA-binding sites as observed by fracture-label. Coating of intact spermatozoa with cationized ferritin results in drastic reduction of the labeling of both protoplasmic and exoplasmic membrane halves. Labeling of sperm cells lysed by short hypotonic shock fails to reveal the presence of WGA-binding sites at the inner surface of the plasma membrane. We conclude that: (a) all WGA-binding glycoconjugates are exposed at the outer surface of the membrane; (b) some of these glycoconjugates correspond to transmembrane glycoproteins that, on fracture, partition with the inner half of the membrane; (c) these transmembrane proteins are accumulated in the region of the plasma membrane that overlies the acrosome; and (d) parallel distribution of intramembrane particles and WGA-binding glycoproteins provides renewed support for the view of particles as the morphological counterpart of integral membrane proteins.     

Splayed Tetrahymena cilia. A system for analyzing sliding and axonemal spoke arrangements

This study makes use of a procedure designed to illustrate, without serial section analysis, the three-dimensional changes in the ciliary axoneme produced by microtubule sliding, and to confirm essential features of the sliding microtubule hypothesis of ciliary movement. Cilia, isolated from Tetrahymena pyriformis by the dibucaine procedure, are attached to polylysine substratum, and treated with Triton X-100. Critical point drying maintains three-dimensional structure without embedding. The detergent removes the membrane and many axonemes unroll, always in an organized fashion so that doublets follow one another in sequence, according to the enantiomorphic form of the cilium. The central pair of microtubules fall to the side as a unit. The parallel doublet microtubules retain relative longitudinal positions in part by interdoublet or nexin links. Spoke organization and tip patterns are preserved in the opened axonemes. We generalize the work of Warner and Satir (Warner, F. D., and P. Satir, 1976. J. Cell Biol. 63:35-63) to show that spoke group arrangements are maintained for all doublets in straight regions, while systematic displacements occur in bent regions. The conclusion that local contraction of microtubles is absent in the axoneme is strengthened, and direct graphic demonstrations of sliding at the ciliary tip are shown. A morphogenetic numbering scheme is presented which results in a quantitative fit of the tip images to the images predicated by the equation for doublet sliding, and which makes possible new comparisons of structural parameters between axonemes and with cilia of other organisms.     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule-based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal-most region of the axoneme, termed the transition zone, which is characterized by Y-shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.     

Identification of UEA I-binding surface glycoproteins of cultured human endothelial cells

Ulex europaeus agglutinin (UEAI) binds mainly to endothelial cells in human tissues. In cultured human umbilical vein endothelial cells TRITC-UEAI gave an even surface staining but no binding to pericellular material. After permeabilization of the cells UEAI decorated the Golgi apparatus as a juxtanuclear structure. Electrophoresis of Triton X-100 lysates of 35S-methionine labeled cells bound to lectin agarose beads showed that a similar set of polypeptides was recognized by UEA-I and WGA while distinctly different polypeptides were bound to LcA-agarose. Surface labelling revealed major glycoproteins with Mr 220 kD, 160 kD, 140 kD, 120 kD, 80 kD and 50 kD, most of which could be extracted with Triton X-100. However, only the 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA 140 kD gp, 120 kD gp and 80 kD gp showed binding to UEA I-lectin. The results show that among a distinct set of surface glycoproteins in cultured human endothelial cells only a few have alpha-l-fucosyl moieties capable of binding to UEAI lectin.     

In vitro phosphorylation of Paramecium axonemes and permeabilized cells

This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.     

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.