An efficient assay for dolichyl phosphate-mannose: protein O-mannosyltransferase.

A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed. The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl [3H]mannose (Dol-P-Man). A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution. The use of LUV allows generation of truly reproducible data and, as an additional benefit, also results in a more than 10 times increase in transfer efficiency. In contrast to the solvent extraction procedures commonly used in previously described PMT assays, the assay reaction product is separated from the radioactive donor substrate on C(18) cartridges. The use of C(18) cartridges allows generation of reproducible data with a low, consistent background and also produces a significant reduction in the time and labor needed for the product workup. In a reaction mixture consisting of 100 microg POPC LUV, 9 x 10(5)cpm (approximately 15 pmol) Dol-P-Man, 100 nmol YNPTSV, and aproximately 4 microg of crude yeast microsomal extract, time-dependent formation of glycosylated product obeys Michaelis-Menten-type kinetics throughout the course of the reaction-until exhaustion of the donor substrate. The linear initial rates of the reaction allowed calculation of an apparent K(m) of 1mM, for the acceptor peptide YNPTSV. Variations in detergent concentration in the assay influence transfer efficiency, possibly through interference with the LUV-based donor substrate delivery system. Hence detergent concentrations should be kept constant.

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.

The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.

The in vitro and in vivo specificity of the family of peptide:N-acetylgalactosaminyltransferases (GalNAcT) is analyzed on the basis of the reactivity and/or inhibitory activity of peptides and protein segments. The transferases appear to be multi-substrate enzymes with extended active sites containing a least nine subsites that interact cooperatively with a linear segment of at least nine amino acid residues on the acceptor polypeptide. Functional acceptor sites are located on the surface of the protein and extended conformations (beta-strand conformation) are preferred. The acceptor specificity of GalNAc-T can be predicted from the primary structure of the acceptor peptide with an accuracy of 70 to 80%. The same GalNAc-T enzymes catalyze the glycosylation of both serine and threonine residues. The higher in vitro catalytic efficiency toward threonine versus serine is the result of enhanced binding as well as increased reaction velocity, both effects being the result of steric interactions between the active site of the enzyme and the methyl group of threonine. Results from substrate binding studies suggest that GalNAc-T catalyzed transfer proceeds via an ordered sequential mechanism.

Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin(9-39).

The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39), we investigated whether the conjugation of a carbohydrate structure to exendin(9-39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9-39) was reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alone or in conjunction with a recombinant GalNAc alpha2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GalNAc or the disaccharide NeuAc alpha2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [125I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.

A scintillation proximity assay for UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase.

A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029-10038) was conjugated to biotin using a di-beta-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 microM acceptor, 0.5 microM nucleotide sugar, and 7.5 U/ml enzyme, the time dependence of product formation obeyed Michaelis-Menten-type kinetics throughout the full course of the reaction-until exhaustion of the donor substrate-and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 +/- 0.12 microM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors.

Organization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase gene and a related processed pseudogene.

We have previously characterized a cDNA that encodes a full length human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) (J.A. Meurer et al., J. Biochem., 118, 568-574, 1995). The present report describes the characterization of the corresponding human GalNAc-transferase gene and a related pseudogene. Two human genomic libraries, lambda and P1, were screened with probes derived from the human GalNAc-transferase nucleotide sequence, resulting in the isolation of four genomic clones. Southern blotting, PCR analysis, and sequencing revealed that three clones, lambda.HG-5, P1.GALN-A, and P1.GALN-B, contained overlapping genomic sequences that encompass over 55 kilobase pairs (kb) of genomic DNA and comprise a portion of the human GalNAc-transferase 5'-and 3'-untranslated regions and the entire coding region. The human GalNAc-transferase gene structure consists of at least 11 exons ranging in size from 99 to > 620 nucleotides which are separated by 10 introns ranging in size from 0.7 to approximately 12.5 kb. The fourth genomic clone, P1-GALN-psi, contained a approximately 2.4 kb sequence region which shares an overall 78.6% nucleotide identity with coding region exons 1 and 3 through 11 of the human GalNAc-transferase gene. However, a lack of intron sequences, as well as the presence of multiple nucleotide mutations, insertions, and deletions that disrupt the potential GalNAc-transferase reading frame, suggest that P1.GALN-psi contains a processed pseudogene. Screening of a human/rodent somatic cell hybrid panel with a P1.GALN-psi probe localized the GalNAc-transferase pseudogene to chromosome 3. Hence, the human genome contains at least two related GalNAc-transferase genes that are located on separate chromosomes.

cDNA cloning, expression, and chromosomal localization of a human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase.

Oligonucleotide primers derived from the cDNA encoding a full-length bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) [Homa, F. L., Hollander, T., Lehman, D. J., Thomsen, D. R., and Elhammer, A. P. (1993) J. Biol. Chem. 268, 12609-12616], were used for PCR to isolate sequences encoding a homologous enzyme from human salivary gland cDNA. Comparison of the human and bovine nucleotide sequences reveals 94.8% sequence identity in their coding regions and 87% identity in their 3-untranslated regions. The translation of the human GalNAc-transferase coding region predicts an amino acid sequence which is nearly identical (99.6%) to that of the bovine counterpart; there are five conservative and one non-conservative amino acid substitutions between the two enzymes. Expression of the bovine and human cDNAs in the insect cell line, Sf9, resulted in the synthesis of proteins which appeared identical on SDS-PAGE and which had similar enzymatic properties. Screening of a somatic cell human/rodent hybrid panel with a probe derived from the human GaLNAc-transferase cDNA sequence indicated that the human GalNAc-transferase gene is localized to chromosome 18.

The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectins.

P- and E-selectins belong to a family of Ca(2+)-dependent lectins and function as receptors for myeloid leukocytes. We have described a panel of monoclonal antibodies which recognize a sialoglycoprotein from human neutrophils and HL-60 promyelocytic cells and inhibit adhesion of these cells to P-selectin. In this study, we show that the E-selectin receptor-globulin (E-selectin Rg) affinity chromatography can isolate specifically only one glycoprotein from [3H]glucosamine-labeled HL-60 cells in a Ca(2+)-dependent manner. This protein has a molecular mass of approximately 120 kDa under reducing conditions, which appears to be identical with the previously characterized glycoprotein ligand for P-selectin. The molecule can be cross-depleted by and cross-bound to the E- and P-selectin columns. The chromatographic profile of desialylated O-linked carbohydrates from molecules purified by P- and E-selectin affinity chromatography are identical. Both have five structures at 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units. PL5 monoclonal antibody to the P-selectin sialoglycoprotein ligand, E-selectin Rg, and antiserum to P-selectin glycoprotein ligand-1 (PSGL-1) all recognize the purified P-selectin ligand on ligand blots and immunoblots. Furthermore, PL5 monoclonal antibody blocks adhesion of HL-60 cells and human neutrophils to E-selectin Rg. Taken together, our results demonstrate that the P- and E-selectin ligand defined in this study is PSGL-1 and suggest that this molecule is an important leukocyte ligand for both P- and E-selectins.

Conversion of a bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase to a soluble, secreted enzyme, and expression in Sf9 cells.

A soluble, secreted UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase, was prepared by substituting the honeybee melittin leader sequence for the sequences coding for the cytoplasmic and membrane spanning domains of a cloned, bovine full-length cDNA (F. L. Homa et al., 1993, J. Biol. Chem. 268, 12609-12616). When this construct was expressed in insect cells using a recombinant baculovirus, a fully active soluble enzyme was recovered from the culture medium. In large-scale preparations, approximately 2-3 mg of enzyme protein was produced per liter of medium. A one-step purification of the soluble molecule on apomucin-Sepharose yielded an essentially homogeneous enzyme preparation. The purified soluble enzyme has a molecular mass of approximately 61 kDa and appears to contain both N- an O-linked oligosaccharides. NH2-terminal sequencing demonstrated that the melittin leader sequence is cleaved at the predicted site and amino acid analysis gave results in close agreement with the composition predicted by the nucleic acid sequence. The enzymatic properties of the soluble, recombinant molecule are similar to those of the enzymes isolated from bovine colostrum and porcine submaxillary gland: the specific activity is approximately 2160 U/mg protein and the Kms for UDP-GalNAc and the synthetic acceptor peptides PPASTSAPG and PPDAASAAPLR are approximately 1.7 microM, 6.5 mM, and 3.6 mM, respectively.

Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

NH2-terminal amino acid sequence obtained from a UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) isolated from bovine colostrum was used for the construction of synthetic oligonucleotide primers. Subsequent polymerase chain reaction and library screenings of a bovine intestine cDNA library produced seven positive clones. The largest clone had a 2294-base pair insert that contained an open reading frame coding for a protein composed of 559 amino acids with a predicted polypeptide molecular mass of 64,173 Da. The cloned molecule has no significant sequence homology to previously reported cloned glycosyltransferases, but appears to have a similar domain structure. It is a type II membrane protein with a 23-amino acid putative transmembrane region starting 8 amino acids from the NH2 terminus. The transmembrane segment of the molecule is immediately followed by a sequence rich in proline residues. The molecule contains three consensus sequences for N-linked glycosylation and five predicted sites for O-glycosylation. Northern blot analysis of poly(A+) mRNA isolated from Madin-Darby bovine kidney cells, bovine mammary tissue, and eight human tissues demonstrated the expression of two transcripts differing in size by approximately 1 kilobase. The cloned DNA was expressed in insect cells using a baculovirus vector. This resulted in an almost 100-fold increase in GalNAc-transferase activity in lysates prepared from cells infected with virus containing the GalNAc-transferase gene compared to cells infected with virus containing DNA coding for an unrelated molecule or uninfected cells. Immunoprecipitation from lysates prepared from infected cells labeled in vivo with [35S] methionine showed a large increase in the recovery of an approximately 67-kDa protein.

The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides.

The acceptor substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) was inferred from the amino acid sequences surrounding 196 O-glycosylation sites extracted from the National Biomedical Research Foundation Protein Database. When analyzed according to the cumulative enzyme specificity model (Poorman, R.A., Tomasselli, A.G., Heinrikson, R.L., and Kézdy, F.J. (1991) J. Biol. Chem. 266, 14554-14561) these data were found to be consistent with an enzymatic active site which interacts with an 8-amino-acid long segment of the substrate, spanning 3 amino acid residues preceding and 4 amino acid residues following the reactive serine or threonine. The model postulates independent interactions of the 8 amino acid moieties with their respective binding sites, designated as subsites P3 through P0 and P1' to P4'. High selectivity is expressed at all subsites toward serine, threonine, and proline. The inferred specificity was confirmed by in vitro bovine colostrum GalNAc-transferase-catalyzed glycosylation of unglycosylated proteins containing predicted sites for O-glycosylation and synthetic peptides designed to be GalNAc acceptors. In synthetic peptides the bovine colostrum GalNAc-transferase glycosylates threonine about 35 times faster than serine. Our results suggest that the specificity of the enzyme is not dependent on any particular secondary structure of the substrate but, rather, it is determined by the amino acids in the acceptor peptide segment as well as by the accessibility of this segment. It also appears likely that bovine colostrum GalNAc-transferase is able to catalyze in vivo the glycosylation of both threonine and serine residues.

Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9.

The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [3H]glucosamine and infected with a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal beta 1-3GalNAc constituting 17 and 66% of the total number of structures, respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked alpha 1-6 to the asparagine-linked N-acetylglucosamine.

Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9.

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.