Cell surface galactosyltransferase: Immunochemical localization

Shaper, Nancy L.; Mann, Paul L.; Shaper, Joël H.

doi:10.1002/jcb.240280305

Cited by 55 publications

(19 citation statements)

References 24 publications

(13 reference statements)

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“…Rabbit anti-rat liver Golgi mannosidase II antibodies (IgG fraction) were prepared and characterized as reported (30). Affinity-purified rabbit anti-bovine galactosyltransferase antibodies were those previously used (31,32). Ovomucoid-gold and fetuin-gold complexes were prepared as reported (33,34 39.5°C for 2 h and then shifted back to 32°C for 30 min, 1 h, or 2 h were also studied.…”

Section: Methodsmentioning

confidence: 99%

DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.

Zuber

Roth

Misteli

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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The temperature-sensitive Chinese hamster ovary (CHO) cell mutant DS28-6 has been previously shown to be pleiotropically defective in protein secretion. We have examined the mutant cells to determine the intracellular site of the block in secretion. By transmission electron microscopy a time-dependent disassembly of the Golgi apparatus was found under nonpermissive temperature, which resulted in the loss of the cisternal stack. Complete reorganization of the Golgi apparatus occurred after shift to permissive temperature. Under nonpermissive temperature, a microtubule-and energydependent redistribution of Golgi mannosidase I and galactosyltransferase into a pattern characteristic of the endoplasmic reticulum (ER) was observed. Inhibition of protein synthesis by cycloheximide had no influence on Golgi mannosidase H redistribution. Evidence for Golgi apparatus-associated processing of oligosaccharides in the ER was obtained by lectin-gold cytochemistry revealing the presence of the galactose (013-+4)N-acetylglucosamine sequence and sialic acid residues. Furthermore, 7-nitrobenz-2-oxa-1,3-diazol-4-yl-tagged ceramide, a lipidic trans-Golgi apparatus marker in CHO cells, exhibited an energy-dependent redistribution into the ER. These effects were fully reversible upon shift to permissive temperature. Thus, mutant DS28-6 cells exhibit key features of the brefeldin A phenotype, which suggests that the observed brefeldin A effects result from interference with a normally occurring cellular process.

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Section: Methodsmentioning

confidence: 99%

DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.

Zuber

Roth

Misteli

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Polyclonal rabbit antisera were prepared against affinity-purified bovine galactosyltransferase isolated from raw skim milk. Affinity-purification of the enzyme (15), characterization of the antisera, and preparation of the monospecific antisera by chromatography on an agarose adsorbent to which galactosyltransferase was covalently linked has been described (5).…”

Section: Methodsmentioning

confidence: 99%

“…Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical (5)(6)(7)(8)(9) and biochemical procedures [comprehensively reviewed in Pierce et al (10) and Shur (11)]. This cell surface distribution has led to the postulate that, in addition to its biosynthetic role, galactosyltransferase has a functional role in intercellular recognition and/or adhesion (12).…”

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confidence: 99%

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Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library.

Shaper¹,

Shaper²,

Meuth³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

176

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A 1.3-kilobase cDNA clone (7A) coding for bovine galactosyltransferase (glycoprotein 4-3-galactosyltransferase, EC 2.4.1.38) was isolated from a Xgtll expression library by immunological screening with monospecific polyclonal antisera to the affinity-purified bovine enzyme. The nucleotide sequence of this clone predicts an open reading frame that starts at the 5' end of the insert and codes for a polypeptide of 334 amino acids with Mr 37,645. Based on a Mr of 57,000 for the membrane-bound enzyme this clone accounts for approximately 61% of the coding sequence. Portions of the predicted amino acid sequence matched the six tryptic peptides isolated from affinity-purified bovine galactosyltransferase. Clone 7A hybridizes to a 4.8-kilobase bovine mRNA and identifies multiple EcoRI restriction fragments in bovine, murine, and human DNA.UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-p-D-galactosyltransferase (glycoprotein 4-/-galactosyltransferase; EC 2.4.1.38) is a Golgi membrane-bound enzyme that participates in the biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids (1, 2). Galactosyltransferase catalyzes the following reaction of UDPgalactose (UDP-Gal) and N-acetylglucosamine (GlcNAc): MnI2 UDP-Gal + GlcNAc Mn Gal(/31-+4)GlcNAc + UDP where the acceptor sugar, N-acetylglucosamine, may be either the free monosaccharide or the nonreducing terminal monosaccharide of a carbohydrate side chain of a glycoprotein or glycolipid (3). Galactosyltransferase can also interact with the regulatory protein a-lactalbumin to form the heterodimer, lactose synthetase (EC 2.4.1.22). This complex catalyzes the transfer of galactose from UDPgalactose to glucose, forming lactose (4). The net result of the specific interaction of galactosyltransferase with a-lactalbumin is to lower the Km for glucose so that lactose synthesis can take place at physiological concentrations of glucose.Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical (5-9) and biochemical procedures [comprehensively reviewed in Pierce et al. (10) and Shur (11)]. This cell surface distribution has led to the postulate that, in addition to its biosynthetic role, galactosyltransferase has a functional role in intercellular recognition and/or adhesion (12). A detailed analysis of the cell surface galactosyltransferase localized to the plasma membrane overlying the intact acrosome of mouse sperm supports a functional role in sperm-egg recognition (7, 13). There is also evidence to suggest that galactosyltransferase might be functionally involved in the T/t complex of the mouse, a region in which mutations lead to abnormal embryonic development and sperm production (14).Because of the multiple functions of this membrane-bound enzyme and our long range goal of determining the relationship between the cell surface and intracellular form of galactosyltransferase, we have taken advantage of molecular cloning techniques to obtain structural information on this enzy...

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“…Primary antibodies were rabbit anti-rat mannosidase II (Moremen and Touster, 1985;Velasco et al, 1993), affinity-purified rabbit antibovine ␤1,4-galactosyltransferase (Shaper et al, 1985), monoclonal anti-DGC, which recognizes a carbohydrate epitope in Drosophila Golgi complex membranes (obtained from V. Malhotra, University of California, San Diego, CA), and monoclonal antiIip31 [Clonab NL2 (Biotest Diagnostics, Denville, NJ), received from T. Nilsson (EMBL)]. Secondary antibodies were Texas red-conjugated goat anti-rabbit immunoglobulin G (IgG) and and goat anti-mouse IgG, and fluorescein-conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA).…”

Section: Indirect Immunofluorescencementioning

confidence: 99%

Cholesterol-independent Targeting of Golgi Membrane Proteins in Insect Cells

Rolls

Marquardt

Kielian

et al. 1997

MBoC

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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.

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Cell surface galactosyltransferase: Immunochemical localization

Cited by 55 publications

References 24 publications

DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.

DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.

Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library.

Cholesterol-independent Targeting of Golgi Membrane Proteins in Insect Cells

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