Spontaneous redistribution of cell-surface glycoproteins in lymphoid cells during cytokinesis.

The 'unperturbed' distribution of plasma membrane glycoproteins during cytokinesis has been examined by immunofluorescence and electron microscopy on dividing mouse and rat lymphoid cells fixed before being labelled with the appropriate reagents. Two groups of molecules which cap 'spontaneously' to the uropod of non-dividing cells, i.e., the common receptors for Helix pomatia (HPA) and peanut agglutinin (PNA) (and in particular the thymocyte glycophorin-like glycoprotein) and membrane immunoglobulins, redistribute spontaneously to the cleavage furrow during cytokinesis. By electron microscopy, the redistributed molecules (HPA receptors) appear to be aggregated in clusters. Other glycoproteins, such as Concanavalin A receptors and Thy.1 antigens, which do not cap spontaneously on interphase cells, remain uniformly distributed or are somewhat depleted over the cleavage furrow. The results suggest that a spontaneous 'transport' of certain membrane molecules from the nuclear pole to the cleavage furrow occurs normally during cytokinesis by a mechanism analogous to that of uropod formation and spontaneous capping in interphase cells. The existence of redistribution phenomena in dividing cells imposes some restrictions on the possible mechanisms of redistribution and on certain aspects of the cleavage process.     

Comparative lectin histochemistry on taste buds in foliate, circumvallate and fungiform papillae of the rabbit tongue.

Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungi-form taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

Lectin ligands on human dendritic cells and identification of a peanut agglutinin positive subset in blood

As only a few cell surface markers for dendritic cells (DC) have been identified to date, this study examined the expression of ligands for lectin on different human DC populations. The ability of Concanavalin A (Con A), Wheat Germ Agglutinin (WGA), peanut agglutinin (PNA), and Helix pomatia (HPA) to bind to cell lines and PBMC and DC populations was analyzed by flow cytometry and specificity of binding confirmed using inhibitory and noninhibitory sugars. The cell lines showed non-lineage-restricted binding with Con A and WGA, independent of sialidase treatment. HPA and PNA bound to a restricted number of lines, but showed broad reactivity after sialidase treatment. The peripheral blood mononuclear cells (PBMC) and directly isolated blood DC, activated CD83(+) blood DC, epidermal Langerhans cells (LC), and monocyte-derived DC (Mo-DC) showed strong binding of Con A and WGA, both before and after sialidase treatment. No HPA binding ligands were detected on PBMC populations, including directly isolated blood DC. Following sialidase treatment CD3(+), CD16(+), and a subset of CD19(+) lymphocytes bound HPA. The lectin PNA bound weakly to CD14(+) monocytes and a subpopulation of circulating DC that were HLA-DR(hi)CDw123 Dr(hi)CDw123(dim)/(neg)CD11c(+). The HLA-DR(mod)CDw123(hi)CD11c(neg) subpopulation did not bind PNA. Without sialidase treatment LC expressed both HPA and PNA ligands, but these were either absent on activated CD83(+) blood DC or weakly expressed on Mo-DC. Following sialidase treatment PBMC populations, activated CD83(+) blood DC, and Mo-DC became PNA positive. Thus human DC express several lectin ligands and PNA binding identifies a subset of blood DC. That may reflect discrete changes associated with stages of DC development or functional maturation.     

Some lectin binding properties of the tongue of the mole rat,Nannospalax xanthodon

We investigated carbohydrate residues on the epithelial surface, in the epithelial cells and in gland cells of the tongue of the mole rat using histochemical methods. We used horseradish peroxidase-conjugated lectins from Helix pomatia (HPA), Arachishypogaea (PNA), Ulexeuropaeus (UEA I), Canavaliaensiformis (Con A). The most intense reactivity was observed in the keratin layer with HPA, UEA I and Con A, and in the epithelial cells with UEA I and Con A. In the glands, we found strong reactivity in serous cells with HPA and Con A, and in mucous cells with HPA and UEA I. PNA did not bind to epithelial or gland cells. Consequently, GlcNAc, fucose and α-D-mannose terminal glycoconjugates are distributed widely; GalNAc terminal glycoconjugates appeared in small amounts.     

Comparative lectin histochemistry on taste buds in foliate,circumvallate and fungiform papillae of the rabbit tongue

Summary Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungiform taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like, paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

The peanut lectin-binding glycoproteins of human epidermal keratinocytes

Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.     

Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoproteins: evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping

Two major rat thymocyte surface glycoproteins, the leucocyte-common (L-C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX-1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing. TRITC-phalloidin staining of the isolated caps demonstrated the presence of F-actin within these structures, and lectin-affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L-C antigen and LSGP caps, but absent from the detergent-insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross-linked glycoproteins with the cytoskeleton during capping.     

Thymic reticulum in mice. IV. The rosette formation between phagocytic cells of the thymic reticulum and cortical type thymocytes is mediated by complement receptor type three

A phagocytic cell of the thymic reticulum (P-TR) with dendritic shape recently has been isolated and characterized. We have previously shown that P-TR have an important role to play in the constitution of the thymic microenvironment. Indeed, P-TR are able to produce interleukin 1 and prostaglandin E2, both of which regulate thymocyte activation and proliferation. They are able also to stimulate the proliferation of syngeneic thymocytes enriched in the medullary type. In the present paper, we analyze a close relationship which exists between P-TR and thymocytes of the cortical type. About 25% of P-TR are able to bind to thymocytes and to form rosettes. Rosetting thymocytes represent about 5% of the total population and are PNA+, Lyt 1+2+, H-2-, and sensitive to in vivo steroid treatment. Pretreatment of P-TR with anti-Mac-1, a monoclonal rat IgG antibody against mouse macrophages and specific for complement receptor type three (CR3), abolished rosette formation. Rosette formation also was found to be inhibited by zymosan-treated serum containing the CR3 ligand, C3bi, and by certain sugars, in particular, N-acetyl-D-galactosamine and L-xylose. Our results suggest that rosetting thymocytes bind to CR3 on the P-TR membrane and that sugar constituents of the carbohydrate moieties on the thymocyte surface may serve as a recognition site during the binding process.     

Distribution of lectin-binding glycosidic residues in the hamster follicular oocytes and their modifications in the zona pellucida after ovulation.

In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohydrate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin-gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progressively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB(4) was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analysis was also performed on labeling by lectin-gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization.     

Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells

Summary The apical surface coat of type II pneumocytes and Clara cells in pre- and post-natal rat lung was examined with lectin histochemical methods. Lectins fromHelix pomatia (HPA), peanut (PNA) andMaclura pomifera (MPA) were conjugated with horseradish peroxidase and used to stain paraffin sections of fixed lung with or without certain pre-treatments. HPA and MPA were observed to react with almost all type II pneumocytes at postnatal day 1. Type II pneumocytes that stained with a sialidase—PNA sequence increased from a few positive cells at postnatal day 5 to many in the adult. It has been reported that the surface coat of type II pneumocytes closely resembles that of Clara cells in its staining with histochemical methods employing cationic dyes or lectins including MPA and PNA. However, staining with HPA, especially after periodic acid oxidation, revealed many type II pneumocytes with strong reactivity but showed only a few Clara cells that were faintly positive. HPA also stained alveolar macrophages. The HPA affinity of macrophages, however, was labile to oxidation with periodic acid or galactose oxidase unlike that of type II pneumocytes. This difference suggests that HPA recognizes more than one type of sugar structure.To whom all correspondence and reprint requests should be addressed.     

Identification of peanut agglutinin-binding glycoproteins on immature human thymocytes

Previous studies in our laboratory have shown that peanut agglutinin (PNA), a lectin specific for the disaccharide Gal beta 3GalNAc, binds to immature (cortical) thymocytes of mouse and man and not to the mature (medullary) cells. Using lectin overlay of protein blots and lectin-affinity chromatography, we have found that the major PNA-binding glycoproteins on total as well as on immature (PNA+) human thymocytes correspond to two bands of Mr 170,000 and 180,000. Another glycoprotein, of Mr 110,000, also binds PNA but to a lesser extent. All three glycoproteins contain sialic acid as demonstrated by cell surface labeling with NaIO4-NaB3H4, binding of wheat germ agglutinin, and reaction with alkaline phosphatase-hydrazide. After treatment with sialidase, binding of PNA to these glycoproteins is significantly enhanced.     

Lectin histochemistry study in the human vas deferens

The oligosaccharide sequences of glycoconjugates in the normal human vas deferens and the nature of the saccharide linkage were studied by lectin histochemistry. The cytoplasm of all epithelial cell types (principal cells, basal cells, and mitochondria-rich cells) and luminal contents reacted positively with WGA, MAA, PNA, DSA, LTA, UEA-I, AAA, and ConA. The reaction was more intense in the stereocilia of principal cells. Cytoplasmic staining was diffuse except for PNA and DSA labeling which was limited to the apical cytoplasm and stereocilia of columnar cells. The cytoplasm of all cell types also reacted diffusely with HPA, although staining was weak and was not observed in the stereocilia. Positive reaction with SBA only was encountered in the stereocilia of principal cells. SNA, LTA, and DBA were unreactive. GNA-labeling showed a granular distribution in the supranuclear cytoplasm of columnar epithelial cells. Reactions with MAA, PNA, DSA, AAA, HPA and SBA disappeared after the -elimination reaction. Reactions with WGA and UEA-I decreased after -elimination or Endo-F digestion. Reactions with ConA and GNA were suppressed by Endo-F digestion. Reactions with PNA, HPA, and SBA increased after desialylation. Of all the lectins that label the luminal contents of the vas deferens, only UEA-I was not found in the luminal contents of seminiferous tubules and epididymis and, thus, this lectin would probably bind to glycoproteins secreted by the vas deferens. The chemical treatments used suggest that this secretion contains fucose residues located in both N- and O-linked oligosaccharides. The other lectins may label secreted proteins, but also structural proteins or proteins reabsorbed from the luminal fluid. The lectin- binding pattern of mitochondria-rich cells in the vas deferens differed from that found in the epididymis.     

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.     

Identification and estimation of the proportion of limiting cell types involved in the phytohemagglutinin response by limiting cell dose-response analysis

Fluoresceinated peanut agglutinin (PNA-FITC) and a monoclonal anti-T-cell antibodies were used to identify human thymocyte subpopulations. The phenotypes of PNA+ and PNA? thymocytes were studied using two different techniques: agglutination with PNA and double-labeling immunofluorescence. The mitogen-responsive PNA- subset was shown to include early thymocytes bearing the OKT9 antigen as well as lymphocytes with the OKT3 phenotype. Nearly all PNA+ thymocytes were found to bind simultaneously OKT4, OKT6, and OKT8 antibodies, whereas about 30% of them weakly bind the OKT3 antibody. These data suggest the existence of several intermediate stages of intrathymic differentiation, including a subset simultaneously bearing the OKT6- and OKT3-defined antigens.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with 35S-methionine-labeled cells showed that HPA recognized two glycoproteins of Mr 170,000 and 400,000 among the secreted products of fibroblasts and two major cellular glycoproteins of Mr 40,000 and Mr 180,000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulse-labeled shortly with 35S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

Proteome analysis of metastatic colorectal cancer cells recognized by the lectin Helix pomatia agglutinin (HPA)

The lectin from Helix pomatia (HPA) binds to adenocarcinomas with a metastatic phenotype but the glycoconjugates of cancer cells that bind to the lectin have yet to be characterized in detail. We used a model of metastatic (HT29) and nonmetastatic (SW480) human colorectal cancer cells and a proteomic approach to identify HPA binding glycoproteins. Cell membrane proteins purified by HPA affinity chromatography, were separated by 2-DE and analyzed by MS. Competitive inhibition experiments with N-acetylgalactosamine, N-acetylglucosamine, and sialic acid confirmed that HPA binding was via a glycan-mediated interaction. Western blot analysis showed that HPA binds to proteins not recognized by an antibody against blood group A epitope. The proteomic study showed the main HPA binding partners include integrin alphav/alpha6 and annexin A2/A4. These proteins were found complexed with microfilament proteins alpha and beta tubulin, actin, and cytokeratins 8 and 18. HPA also bound to Hsp70, Hsp90, TRAP-1, and tumor rejection factor 1. This study revealed that the prognostic utility of HPA lies in its ability to bind simultaneously to many glycoproteins involved in cell migration and signaling, in addition, the proteins recognized by HPA are glycosylated with structures distinct from the blood group A epitope.     

Identification of peanut agglutinin receptors related to the state of tumoral liver cell differentiation.

The relationship between cell differentiation/tumorisation and plasma membrane glycoproteins was approached using peanut agglutinin (PNA) a lectin specific for the Gal-beta(1,3)GalNAc sequence and a homologous cell system consisted of normal rat hepatocytes (HyC) and a poorly differentiated hepatoma (ZHC). This work is focused on the molecular nature of PNA receptors. PNA bound strongly to ZHC, but bound very weakly, if at all to hepatocytes. After sialidase treatment this binding was slightly enhanced in ZHC and HyC. The total number of binding sites on ZHC was 9.6 x 10(6)/cell and 1.2 x 10(7)/cell before and after sialidase treatment respectively. In contrast, this number could not be calculated on HyC, even after sialidase treatment. The PNA receptors were isolated and identified from ZHC using affinity chromatography on immobilized PNA and lectin overlay. Two bands were revealed after SDS-PAGE of PNA receptors: a major one with a relative molecular mass of 160 kDa and a minor one of 110 kDa. The latter disappeared after sialidase treatment of ZHC suggesting the possibility that these two bands could be less and more sialylated forms of the PNA receptors, respectively. In contrast no PNA receptors could be detected on HyC. These PNA receptors could be considered O-linked glycoproteins containing the Gal-beta(1,3)GalNAc disaccharide because: i) PNA carbohydrate specificity toward this disaccharide found in this glycoprotein type; ii) their carbohydrate composition with Gal and GalNAc but not man residues; iii) their sensitivity to alkaline treatment; and iv) strong inhibition of PNA binding to ZHC with the Gal-beta(1,3)GalNAc structure. The absence of PNA receptors on HyC appeared to be related to the absence of this glycoprotein containing the disaccharide but not to the change or failure of glycosylation of the polypeptide chain of PNA receptors. The relationship between the presence of PNA receptors and differentiation/tumorisation phenomena as well as the mechanism that induced the expression of these receptors are discussed.     

Leukemic transformation in hrs mice occurs in an immature-nonactivated thymocyte subpopulation

The murine leukemic strain HRS/J has an autosomal-recessive, mutant gene, hr, with homozygotes (hr/hr) having a 72% incidence of thymic leukemia at 18 months of age compared to 20% in heterozygotes (hr/+). This study was done to (a) determine if expression of thymocyte differentiation and murine leukemia virus (MuLV) antigens during leukemic transformation were different in hr/hr compared to hr/+ mice, (b) define the subpopulations that were targets for leukemic transformation, and (c) compare the results to reports in other leukemic strains. Flow cytometry analysis of thymus cell suspensions was done with anti-T-cell and anti-H-2 monoclonal antibodies, peanut agglutinin (PNA), and heteroantisera to MuLV antigens. Thymocytes of 1- to 3-month-old HRS/J mice were Thy 1.2+, Lyt 1+2+, H-2Kk-, and MuLV- with an immature-nonactivated phenotype, i.e., PNA+, and Iak-. Preleukemic and leukemic thymocytes showed diversity in expression of Thy 1.2 and Ly antigens with increased H-2Kk and MuLV expression. No differences in phenotype patterns were noted between hr/+ and hr/hr mice during the time course of leukemogenesis. Persistently high PNA/low Iak expression of preleukemic and leukemic thymocytes indicated that the target for HRS leukemic transformation was an immature-nonactivated thymocyte subpopulation in contrast to AKR/J mice in which leukemic transformation involves a mature-activated thymocyte subpopulation. These findings suggest that spontaneously generated leukemogenic viruses in HRS mice have tropism for thymocytes of an immature-nonactivated phenotype.