Spermatozoa from a marsupial, the brushtail possum, contain beta1,4-galactosyltransferase.

Beta1,4-galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm-egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of beta1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall beta1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P<0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.

Cholesterol-independent targeting of Golgi membrane proteins in insect cells.

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by confocal double immunofluorescence microscopy.

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.

Light microscopic localization of glycosyltransferase activities in cells and tissues.

We describe an assay for light microscopic visualization of specific glycosyltransferases on tissue sections or on cells. The assay uses a sequence of enzyme reactions that yields two moles of NADH for each mole of the uridine-5'-diphosphate (UDP) released during transfer of a monosaccharide from a UDP sugar to an acceptor. When diaphorase and tetrazolium salts are present in the incubation mixture, the tetrazolium salts are reduced to colored diformazans, which precipitate at the sites of glycosyltransferase activity. The validity of the assay was established by applying the technique to spermatozoa and liver, in which some glycosyltransferases have previously been localized. When suspensions of mouse spermatozoa were assayed for galactosyltransferase (GalTase) activity, diformazan precipitates appeared on the plasma membranes overlying the anterior heads of the spermatozoa, in agreement with immunochemical localizations. In mouse liver slices assayed with bilirubin as acceptor for glucuronyltransferase (GluTase) activity, dense diformazan deposits appeared on the hepatocytes but not on endothelial cells, also in agreement with immunochemical data. In the absence of acceptor or UDP sugar donor, diformazan deposits were minimal and random in all tissues tested. The assay's versatility was tested by incubating tissues with different sugar donors and acceptors to localize other sites of transferase activity. In mouse frozen liver sections, GalTase activity occurred in both hepatocytes and endothelial cells; in sections of rat submaxillary glands, GalTase activity was detected in mast cells. In liver sections, GlcuTase activity with o-aminophenol as acceptor was located primarily on the endothelial cells. With the appropriate sugar donor and acceptor, this assay should detect any transferase, other than the glucosyltransferases, that utilizes UDP sugars.

Subcellular localization of N-acetylglucosaminide beta 1----4 galactosyltransferase revealed by immunoelectron microscopy.

We prepared a monoclonal antibody (MAb) against N-acetylglucosaminide beta 1----4 galactosyltransferase purified from F9 embryonal carcinoma cells. The MAb recognized the protein portion of the enzyme, since it inhibited galactosyltransferase activity, reacted with the enzyme both from F9 cells and from bovine milk, and did not exhibit anti-carbohydrate activity. Using this MAb, we studied the subcellular localization of the enzyme by immunoelectron microscopy. Intense staining was observed in trans-Golgi stacks within testicular interstitial cells and mucous neck cells, confirming the specificity of the immunological reaction. Cell surface galactosyltransferase was detected in the following regions: cultured cells such as F9 embryonal carcinoma cells, testicular interstitial cells, seminiferous tubule epithelial cells, Sertoli cells, the head of the epididymal sperm, epididymal epithelial cells, and apical surfaces of epithelial cells in the fundic gland and of intestinal goblet cells. The use of Triton X-100 intensified the cell surface immunoreactivity, and in certain cases the mode of distribution of the cell surface enzyme was different from that described in previous reports. In addition, nuclear envelopes of cultured cells were distinctly stained. The possible significance of the latter finding is discussed in relation to recent advances in nuclear localization of glycoproteins.

An immunohistochemical study of beta1,4-galactosyltransferase in human skin tissue.

An immunohistochemical investigation of beta1,4-galactosyltransferase (beta1,4-GalT) on human skin tissue was performed on formalin-fixed paraffin-embedded sections using a monoclonal antibody, MAb8628, which specifically recognizes a protein moiety of human beta1,4-GalT. Distribution of the galactose beta1,4-N-acetylglucosamine (Gal beta1,4GlcNAc)-R epitope was also detected by staining with Ricinus communis agglutinin (RCA) 120. The beta1,4-GalT was observed to be localized at the perinuclear region of epidermal keratinocytes. The fine localization was also observed at the supranuclear region in the cells of apocrine glands, eccrine ducts and glands. The positive staining with RCA 120 was well colocalized with the cells expressing the beta1,4-GalT. An electron microscopic study revealed that positive signals of beta1,4-GalT definitely reside in the Golgi apparatus. No immunoreactivity was observed in any other intracellular structure or on the cell surface. These findings strongly indicated that the beta1,4-GalT is the major enzyme responsible for the Gal beta1,4GlcNAc-R epitope synthesis in human skin tissue.

Immunohistochemical localization of beta-1,4-galactosyltransferase in human pancreatic tissues.

beta-1,4-Galactosyltransferase (GalTase) is the glycosyltransferase in the Golgi apparatus that transfers galactose from UDP-galactose to terminal N-acetylglucosamine residues in glycoconjugates with formation of a beta-1,4 linkage. Neoplasms undergo various changes in the carbohydrate moieties of their glycoconjugates. This process also indicates the possibility of changes in glycosyltransferases themselves. Therefore, we compared the binding pattern of a monoclonal antibody (MAb8628) against GalTase in both normal and neoplastic exocrine pancreatic tissues. Ten normal and 11 neoplastic human exocrine pancreatic tissues obtained from surgery were used. Frozen sections were incubated with this antibody. Supranuclear regions and terminal bars of normal duct cells and acinar cells revealed positive staining for GalTase at the light microscopic level. Centroacinar cells revealed positive staining in their perinuclear region. Neoplastic cells were also stained in their supranuclear regions and terminal bars. Supranuclear regions were well developed in neoplastic cells and intensely stained compared with those in normal cells. The supranuclear regions and the terminal bars corresponded to the trans cisternae of the Golgi apparatus and the junctional complex (i.e., tight junction and adherens junction), respectively, seen at the electron microscopic level. Pancreatic neoplastic changes thus led to an increase in the expression of GalTase in the Golgi apparatus, the increase of which may have an important effect on the intercellular adhesion and communication among pancreatic epithelial cells. Measurement of this enzyme is useful for diagnosis of exocrine pancreatic neoplastic changes from normal tissues.

Peripheral B lymphocyte beta1,3-galactosyltransferase and chaperone expression in immunoglobulin A nephropathy.

PURPOSE: Aberrant O-glycosylation of serum IgA(1) is presumed to be one of the main pathogenesis of immunoglobulin A nephropathy (IgAN). beta1,3-galactosyltransferase (beta1,3GT), whose activity requires coexistence of a specific chaperone, is the main enzyme which participate in the glycosylation process. The current study was carried out to elucidate the expression level of beta1,3GT (C1GALT1) and its chaperone (Cosmc) in IgAN, and their relationships with clinical features as well as IgA glycosylation level. DESIGN, SETTING AND SUBJECTS: Forty-one patients with IgAN, 21 patients with non-IgAN glomerulonephritis and 26 normal controls were included in the present study. Peripheral B lymphocytes were isolated, and then expression level of C1GALT1 and Cosmc were quantitatively measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Serum IgA level and glycosylation level were determined by enzyme-linked immunosorbent assay (ELISA) and VV lectin-binding method. Correlation analysis was performed between C1GALT1/Cosmc expression levels and clinical manifestations (severe proteinuria, renal dysfunction, gross haematuria). RESULTS: B-lymphocyte Cosmc gene expression level was significantly lower in IgAN patients than that of normal control and non-IgAN patients (P<0.05), whilst no apparent disparity was observed in C1GALT1 expression level. Cosmc expression showed a negative correlation with IgA O-glycosylation level indicated by VV lectin-binding assay. Statistical analysis also indicated that the level of Cosmc expression was negatively correlated with severe proteinuria (P<0.05) instead of gross haematuria (P>0.05). CONCLUSION: These data suggested that the aberrant IgA O-glycosylation in IgAN was resulted from a downregulation of beta1,3GT chaperone (Cosmc) expression in B lymphocyte, which is closely associated with clinical characteristics of the disease. This downregulation might be one of the fundamental pathogenic abnormalities in IgAN.

An ELISA-based assay for detergent-solubilized cellular beta 1,4-galactosyltransferase activity. Use of a polyacrylamide derivative with GlcNAc-beta side chains as a solid phase acceptor substrate.

In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcbeta side chains, termed PAP(GlcNAcbeta), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble beta1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0 approximately 5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GaIT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcbeta) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.

Mitotic Golgi clusters are not tubular endosomes.

HeLa cells were incubated with 15 nm BSA-gold for 1 or 2 hours to mark the endocytic pathway and mitotic cells were then isolated by shake-off. Thin, frozen sections were labelled with antibodies against two resident Golgi markers, beta-(1,4)-galactosyltransferase and N-acetylglucosaminyltransferase I. Detection of the latter was aided by the use of a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. The secondary antibodies were coupled to either 5 or 10 nm gold so that the distribution of each of the three markers could be followed. Qualitative and quantitative studies showed that there were two populations of clusters, those described by us earlier and termed Golgi clusters (Lucocq et al. (1987) J. Cell Biol. 104, 865-874), containing either or both Golgi markers, and clusters of tubular endosomes containing BSA-gold. There was very little overlap showing that Golgi clusters cannot be tubular endosomes as concluded by Tooze and Hollinshead (1992) Eur. J. Cell Biol. 58, 228-242.

Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay.

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.

[Induction of N-acetyllactosamine synthetase activity in the mouse mammary gland by prolactin and placental lactogen hormone]. / Induction d'une activité N. acétyl lactosamine synthétase dans la glande mammaire de souris par la prolactine et l'hormone lactogène placentaire

The authors show that the ovine prolactine promote induction of N. acetyl lactosamine synthetase in tissue culture of mammary glands of pregnant mice. A crude extract of human placenta has also a lactogenic activity as tested by the same method, but in this case the blank values are very high for large concentration of crude extract. The molecular forms of HCS are tested: the slow band has a lactogenic activity, the intermediate band has no activity and the rapid band seems to be inhibitory.

Immunocytochemical localization of the protein reactive to human beta-1, 4-galactosyltransferase antibodies during chick embryonic skin differentiation.

BACKGROUND: beta-1, 4-Galactosyltransferase (GalTase) transfers galactose from UDP-galactose to terminal N-acetylglucosamine in glycoconjugates and is located both in the Golgi apparatus and in the plasma membrane. The cell surface GalTase is thought to be involved in cell-to-cell recognition and cell-to-extracellular matrix interaction. METHODS: By the use of specific monoclonal antibodies against human GalTase, changes in cell surface localization of the protein reactive to the antibodies in chick embryonic skin during its differentiation in vivo and in vitro were detected immunohistochemically at both light- and electron microscopic levels. The distribution of glycoconjugates having terminal N-acetylglucosamine residues was detected by staining with succinylated wheat germ agglutinin (s-WGA). RESULTS: Under the light microscope, intense immunostaining was observed in the keratinized epidermis, particularly in the intermediate layer. Marked changes in the localization of the staining were observed in vitamin A-induced mucus-secreting skin, in which keratinization was suppressed. The localization of the immunostaining was in parallel with that of glycoconjugates having terminal N-acetylglucosamine residues. Immunoelectron microscopically the immunostaining was located on the cell surface and in the intercellular space of the desmosomes in the intermediate cells of the keratinized epidermis. However, the staining was not present on the cell surface but was detected on the limiting membrane of the mucous granules, in the mucous metaplastic epidermis. In contrast, the staining was always found in the Golgi apparatus in all of the cells. CONCLUSIONS: These results suggest that the protein reactive to human GalTase antibody may be involved in chick epidermal differentiation.

Can relative spermatozoal galactosyltransferase activity be predictive of dairy bull fertility?

The best and poorest bovine semen samples used commercially for artificial insemination in dairy cattle typically differ in pregnancy rates by 20 to 25% but are within a range that pregnancy rates cannot be predicted consistently by commonly used laboratory assays. Sperm motility and morphology are the characteristics most often evaluated. Laboratory assays that measure other functional traits of sperm may be useful as supplemental assays to increase the reliability of predicting fertility. One such functional trait is the ability of sperm to bind to the zona pellucida, a process mediated by complementary receptors on each gamete. On mouse sperm, beta1,4-galactosyltransferase acts as a receptor for the zona pellucida. Beta1,4-galactosyltransferase is expressed on sperm from many mammals, including bovine sperm, and is a candidate for a zona pellucida receptor. The ability of sperm to bind to the zona pellucida may be related to the amount of beta1,4-galactosyltransferase present on sperm. The aim of this work was to determine if bull sperm beta1,4-galactosyltransferase activity was related to fertility. Beta1,4-galactosyltransferase enzyme assays were performed on sperm from 24 bulls whose fertility was estimated by nonreturn rate and on sperm from a second group of seven bulls whose fertility was ranked by in vivo competitive fertilization. Beta1,4-galactosyltransferase activity varied between individual bulls but was not correlated to fertility as estimated by nonreturn rate or by competitive fertilization. These results demonstrate that beta1,4-galactosyltransferase activity on sperm varies between animals, but that beta1,4-galactosyltransferase activity alone is not an accurate indicator of fertility in dairy bulls.

Excess vitamin A decreases the specific activity of galactosyltransferase in Golgi apparatus of rat liver.

This study investigated the effects of vitamin A excess on hepatic galactosyltransferase (EC 2.4.1.13) activity in livers of rats achieved either by feeding of high levels of retinyl palmitate for 16 wk or gavaging with retinol in olive oil for 3 d. Both hypervitaminotic conditions were characterized by hepatic lipid accumulation. Golgi apparatus fractions were isolated and purity of the fractions was monitored by marker-enzyme analyses and electron microscopy. The quality of the fractions isolated from livers of rats receiving vitamin A excess was not different from that of fractions from control rats. An increase in fat-storing cells in liver, observed in vitamin A excess, coincided with the presence of a floating lipid layer present during isolation of the Golgi apparatus. Galactosyltransferase specific activity (with ovomucoid as acceptor) of Golgi apparatus of rats fed excess vitamin A was 27% of control with chronic feeding and 59% of control with administration by gavage. Activity of another luminally oriented protein, uridine 5'-diphosphate phosphatase, was increased under both in vivo regimens. Vitamin A content of Golgi apparatus, as determined by high performance liquid chromatography, correlated negatively with galactosyltransferase activity after both chronic and acute administration of excess vitamin A.

Role of cell membrane galactosyltransferase in concanavalin A agglutination of erythrocytes.

It has been previously observed that rabbit erythrocyte cell surface galactosyltransferase appears to play a role in concanavalin A agglutination of these erythrocytes (Podolsky et al., 1974). Further, a correlation between the occurrence or level of cell surface galactosyltransferase and concanavalin A agglutinability of other cell types has also been observed. The mechanism by which rabbit erythrocyte galactosyltransferase participates in concanavalin A agglutination has now been further defined. The enzyme was solubilized and purified. Characterization of the enzyme properties has shown them to be similar to those reported for other purified galactosyltransferases. Amino acid and carbohydrate analysis showed a high asparagine content and the presence of D-mannose. Specific alpha-mannosidase treatment of the enzyme showed that some of these D-mannose residues were terminal sugars. The purified enzyme also conferred concanavalin A agglutinability to non-agglutinable human erythrocytes. However, the ability to confer concanavalin A agglutinability was unrelated to the enzyme activity per se (as measured with fetuin acceptor) but appeared to be entirely dependent on the presence of terminal alpha-linked D-mannosyl residues in the enzyme structure. These findings suggest that the presence of terminal alpha-mannosidyl residues on cell surface glycoproteins such as galactosyltransferase may be the determining factor in agglutination of cells by concanavalin A.

Isolation of Golgi membranes from tissues and cells by differential and density gradient centrifugation.

This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran-treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self-generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self-generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.