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L A Tabak

Publications and source records attributed to L A Tabak.

At least 37 records · Page 2Linked to original sources
PubMed1996

Charge distribution of flanking amino acids influences O-glycan acquisition in vivo.

The elements that regulate O-glycosylation are poorly understood. We have developed a novel in vivo system to analyze the role of flanking sequence on the modification of a single well characterized O-glycosylation site derived from human von Willebrand factor (PHMAQVTVGPGL). A secreted chimeric reporter protein, containing the human von Willebrand factor sequence, an antibody recognition epitope, and a heart muscle kinase site, was engineered and expressed in COS7 and MCF-7 cells. Glycosylated and non-glycosylated forms of the immunoprecipitated reporter were resolved electrophoretically and their relative amounts quantitated. Using mutational analysis we find that the glycosylation apparatus of COS7 cells can accommodate a broad range of changes in the flanking sequence without compromising glycosylation, but that the distribution of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to the glycosylation site being particularly sensitive. A combination of acidic residues at positions -1 and +3 almost completely eliminates glycosylation of the reporter in both COS7 and MCF-7 cells. The overall density of charged amino acids is less important since substitution of acidic residues at position -2, +1, and +2 had no effect in the level of glycosylation observed.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of the gene encoding rat submandibular gland apomucin, Mucsmg.

Mucin glycoproteins are a major constituent of salivary secretions and play a primary role in the protection of the oral cavity. Rat submandibular glands (RSMG) synthesize and secrete a low molecular weight (114 kDa) mucin glycoprotein. We have isolated, partially sequenced, and characterized the gene which encodes the RSMG apomucin. The gene is encoded by three exons of 106 nt, 69 nt, and 991 nt, separated by introns of 921 nt and 12.5 kb. CAAT and TATA elements are present, at -68 and -26, respectively, in the 5' flanking sequence of the RSMG apomucin gene. The tandem repeat domain present in exon III consists of ten tandem repeats of 39 nt encoding the consensus sequence PTTDSTTPAPTTK. Sequence comparison and organization of the nucleic acid sequence encoding the tandem repeats of two alleles for this gene suggests that the apomucin gene has undergone recombinational events during its evolution. No significant sequence similarity was found with other mucin genes, or with other known salivary gland-specific genes. The gene was localized to rat chromosome 14 using somatic cell hybrids that segregate rat chromosomes. Since this, to our knowledge, represents the first RSMG mucin gene cloned, we have designated this gene Mucsmg.

Amino Acid Sequence↗
PubMed1996

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase using synthetic glycopeptide substrates.

The factors determining glycosylation of mucin type glycoproteins are not well understood. In the present work, we investigated the role of the peptide moiety and of the presence of O-glycan chains on O-glycosylation by UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyl-transferase (ppGalNAc-T). We used purified ppGalNAc-T from bovine colostrum and a series of synthetic glycopeptide and peptide substrates most of which contained sequences derived from the tandem repeat region of MUC2 mucin. The rate of incorporation of GalNAc into Thr was significantly greater than toward Ser residues. The presence of one or two GalNAc-Thr moieties in the substrate significantly reduced enzyme activity, and this effect was more pronounced when the disaccharide Gal beta 1-3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal beta 1-3GalNAc-Thr occurs at a slower rate than primary glycosylation of carbohydrate-free peptide. Analysis of products by HPLC showed that the enzyme was selective in glycosylating peptides or glycopeptides with the PTTTPIST sequence in that the preferred primary glycosylation site was the third Thr from the amino-terminal end; secondary glycosylation depended on the site of the primary glycosylation. Negatively but not positively charged amino acids on the carboxy-terminal side of the putative secondary glycosylation site resulted in high activity suggesting charge-charge interactions of substrates with the enzyme. These studies indicate that O-glycosylation by bovine colostrum ppGalNAc-T is a selective process dependent on both the amino acid sequence and prior glycosylation of peptide substrates.

Animals↗
PubMed1995

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

Alleles↗
PubMed1995

Kinetic analysis of a recombinant UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase.

A mammalian expression vector was designed to express a secreted soluble form of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) with a metal binding site (HHWHHH) at the NH2 terminus. The recombinant enzyme was purified to homogeneity from COS-7 cell media by sequential chromatography on columns of NiCl2-chelating Sepharose, Affi-Gel blue, and Sephacryl S-100. Kinetic parameters of recombinant and native polypeptide GalNAc transferase were comparable for the donor UDP-GalNAc and for the peptide acceptor AcTPPP, EPO-T (PPDAATAAPLR), and HVF (PHMAQVTVGPGL). Initial velocity and product inhibition studies were carried out with purified recombinant polypeptide GalNAc transferase and the substrates UDP-GalNAc and peptide EPO-T. Initial velocity data was consistent with a sequential type mechanism in which binding of both substrates precedes product release. Product inhibition analysis using UDP showed competitive inhibition against UDP-GalNAc and a noncompetitive inhibition against peptide EPO-T. The dead end peptide analogue EPO-G (PPDAAGAAPLR) was a noncompetitive inhibitor of UDP-GalNAc and a competitive inhibitor of peptide EPO-T. Collectively, the results suggest that the most probable kinetic mechanism for the enzyme is one in which both substrates must bind in a random order prior to catalysis. Interestingly, the Km for EPO-T is similar to the Ki for EPO-G, suggesting that peptide interaction with the polypeptide GalNAc transferase does not require a hydroxyamino acid.

Amino Acid Sequence↗
PubMed1995

Cloning and sequence homology of a rat UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) cDNA was amplified from rat sublingual, submandibular and parotid glands, brain, skeletal muscle, and liver, using the polymerase chain reaction (PCR) and sequences derived from bovine polypeptide GalNAc transferase-Type 1 (polypeptide GalNAc transferase-T1). The transcripts encoding the rat sublingual gland and bovine enzymes were 91% identical in nucleotide sequence, except in their 5' and 3' untranslated regions. The enzymes encoded by the rat and bovine cDNAs were 559 amino acids in length and were virtually identical (98% amino acid sequence identity and 99.5% homologous overall). Northern blot analysis indicates that the polypeptide GalNAc transferase-T1 transcripts are expressed in many tissues but at widely differing levels. Although the amino acid sequence of polypeptide GalNAc transferase-T1 is conserved among mammals, the pattern of tissue expression varies between rats and humans. For example, the steady-state level of polypeptide GalNAc transferase-T1 transcript is quite low in lung relative to other rat tissues, whereas high expression of this transcript is detected in human lung. Therefore, we surmise that isoforms of polypeptide GalNAc transferase must exist and that isoforms are expressed in a tissue-dependent fashion. Searches of the GenBank database have revealed homologous sequences for several isoforms derived from several human tissues. In addition, hypothetical proteins from C. elegans also display strong homology; evidence suggests six ancestral isoforms of polypeptide GalNAc transferases may exist in C. elegans.

Amino Acid Sequence↗
PubMed1995

Expression of a functional rat salivary cystatin S polypeptide in Escherichia coli.

Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult. A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned. A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector. The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromatography on a MonoQ column. The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S. The recombinant protein exhibited papain inhibition activity comparable to natural cystatin. This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E. coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor.

Amino Acid Sequence↗
PubMed1995

Facilitated DNA transfer to rat submandibular gland in vivo and GRP-Ca gene regulation.

The internalization of DNA can be facilitated by adenovirus infection. Using the replication-deficient adenovirus, Ad-dl312, and a plasmid-based firefly luciferase gene as a reporter, we have optimized the uptake and expression of DNA in rat submandibular glands in vivo. Luciferase expression is transient and peaked at approximately 18 h after infection. Luciferase activity increased with plasmid concentration and was greatest at 10(9) to 10(10) plaque-forming units of Ad-dl312 per gland. We next examined the expression in vivo of plasmids containing deletions of the glutamine/glutamic acid-rich protein (GRP-Ca isoform) gene upstream region linked to a chloramphenicol acetyltransferase (CAT) reporter. Constructs with 9.4, 6.3, and 2.7 kb and 17 base pairs of upstream sequence gave relative CAT activities of 100, 30, 7.6, and 38.5, respectively. With the 9.4-kb GRP-Ca construct, CAT was preferentially expressed in acinar cells, which is characteristic of GRP. This gene transfer approach should prove useful in the further study of gene expression in salivary glands and other organs.

Adenoviridae↗
PubMed1995

Characterization of low-molecular-weight peptides in human parotid saliva.

The low-molecular-weight components of human saliva remain poorly characterized. Therefore, low-molecular-weight peptides (Mr < 3000) have been purified from human parotid saliva and characterized with respect to their amino acid sequence. From the sequences obtained, it is likely that these peptides are derived from proteolysis of the hydroxyapatite-interactive human salivary proteins, histatins, proline-rich proteins, and statherins. Since human parotid saliva is an amicrobial fluid, much of the low-molecular-weight peptide fraction of this secretion appears to be derived from the proteolytic processing of the larger proteins. Because of their small size, these peptides are likely to be in exchange with dental plaque fluid and may therefore help modulate events such as demineralization/remineralization, microbial attachment, and dental plaque metabolism at the tooth-saliva interface.

Adult↗
PubMed1995

Association of free arginine and lysine concentrations in human parotid saliva with caries experience.

We determined the free-amino acid content of stimulated parotid (ductal) saliva from two groups of adult subjects whose caries experiences were markedly different. The levels of free arginine and free lysine in the parotid saliva of caries-free adults were significantly higher than those found in the parotid saliva of individuals with a history of dental decay. There was no correlation, however, between the levels of these amino acids and the DMFS score within the caries-susceptible groups. Microbial catabolism of dibasic amino acids contributes to the neutralization of plaque acids and may partially account for the higher resting plaque pH observed in caries-free subjects. Alternatively, the elevations observed in free levels of arginine and lysine may reflect a systemic alteration in amino acid metabolism which is common to the caries-free group of subjects.

Adolescent↗
PubMed1994

Molecular cloning of a rat submandibular gland apomucin.

Overlapping cDNA clones which encode the protein core of a rat submandibular gland mucin-glycoprotein have been isolated and characterized. Sequence analysis revealed a translated region of 966 nucleotides encoding a protein of 322 amino acid residues. The translational start site begins with a putative signal sequence comprising the initial 22 N-terminal residues. The predicted secreted portion of the apomucin revealed three distinct domains: an N-terminal domain which is enriched in glutamine (14%), proline (13%), and tyrosine (10%); a central region which consisted of eleven, 39-base pair tandem repeats with the consensus sequence PTTDSTTPAPTTK; and a C-terminal domain which is enriched in threonine and serine residues (47%) which are not part of a repeat motif. The expression of apomucin transcript appears restricted to the rat submandibular and sublingual glands. Southern blot analysis of rat genomic DNAs suggested a low copy number (1, 2) for this apomucin gene and a limited polymorphism in the number of tandem repeats. Collectively, our sequence and expression data indicate that the cloned rat submandibular gland apomucin is distinct from any of the other salivary (bovine, porcine, or human) or rat apomucins reported thus far.

ABO Blood-Group System↗
PubMed1994

Examination of rat salivary glands for the presence of the aquaporin CHIP.

The aquaporin CHIP, AQP-CHIP, is a 28-kDa integral membrane protein that functions as a water channel in kidney and red blood cells. We have examined rat salivary glands for the presence of this molecule. In Northern blot analyses an AQP-CHIP cDNA hybridized to rat kidney mRNA approximately 20-fold more than to mRNA from the rat parotid gland. Western blot analyses, using antisera against human AQP-CHIP, showed that AQP-CHIP was present in crude membranes from rat salivary glands to a much lesser extent than in rat kidney membranes. A 2.8 kb cDNA encoding AQP-CHIP was also isolated from an adult rat parotid gland cDNA library. Immunolocalization studies using thin and ultrathin cryosections and light and electron microscopy, however, revealed that no glandular epithelium was labeled. In contrast, significant immunolabeling was present in microvascular endothelial cells. Non-fenestrated endothelia of capillaries and venules exhibited labeling on both their apical and basolateral membranes. The present study demonstrates that AQP-CHIP is not involved directly in fluid secretion from salivary epithelial cells. Further, this study provides strong evidence that AQP-CHIP is an endothelial cell water channel found within salivary gland tissue which may contribute to water permeability in the capillary beds.

Animals↗
PubMed1994

The effect of milk and casein proteins on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite.

Experiments sought to determine the nature of the binding of milk proteins to hydroxyapatite (HA) and to saliva-coated hydroxyapatite (sHA), and to determine the effect of milk and casein on the adherence of Streptococcus mutans GS-5 to sHA. The binding of radiolabelled alpha-casein to HA was reduced when incubated simultaneously with parotid saliva, and enhanced in the presence of milk. The binding of beta- and kappa-casein to HA was unaffected by the presence of parotid saliva and enhanced by the presence of milk. The in vitro bacterial adherence of Strep. mutans GS-5 to sHA beads was reduced when beads were coated with milk instead of buffer, or when bacteria were added to sHA in the presence of milk instead of buffer. Casein proteins (alpha, beta, kappa) added to sHA simultaneously with bacteria inhibited the adherence of Strep. mutans GS-5 to sHA. kappa-Casein, when bound to sHA, inhibited streptococcal adherence to sHA; alpha- and beta-casein, when bound to sHA, had no effect on streptococcal adherence. Fractionation of kappa-casein by anion-exchange chromatography revealed the anti-adherence activity of kappa-casein was mediated primarily by a 40,000 mol. wt. glycoprotein-containing fraction. These data show that milk, particularly kappa-casein fractions, can modulate the adherence of Strep. mutans GS-5 to SHA surfaces in vitro.

Adult↗
PubMed1993

Purification, cloning, and expression of a bovine UDP-GalNAc: polypeptide N-acetyl-galactosaminyltransferase.

Partial amino acid sequence was obtained from UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) purified from bovine colostrum. Oligonucleotide primers designed from these sequences were used to amplify and clone a polypeptide GalNAc transferase cDNA from bovine placental mRNA. The cDNA encodes an open reading frame, which is 519 amino acids in length and contains the predicted N-terminal and internal amino acid sequence derived from three Lys-C peptides obtained from the purified protein. There was no sequence homology with the UDP-GalNAc: Fuc alpha 1,2Gal alpha 1,3GalNAc transferase. To verify the authenticity of the clone, the cDNA was cloned in frame with an insulin secretion sequence and was expressed transiently in COS-7 cells. Polypeptide GalNAc transferase activity was detected in the culture medium; no activity was detected in the media of mock-transfected cells. Previous studies have shown that the polypeptide GalNAc transferase from bovine colostrum glycosylates threonine residues more efficiently than serine residues in the same peptide context (O'Connell, B. C., Hagen, F. K., and Tabak, L. A. (1992) J. Biol. Chem. 267, 25010-25018). We found that the cloned polypeptide GalNAc transferase glycosylates the threonine-containing peptide, PPDAATAAPLR, at a 58-fold greater rate than the serine-containing homologue, PPDAASAAPLR. The ratio of the in vitro threonine and serine glycosylation rate is identical for the cloned placental and purified colostral enzymes. It is not known if the preference for threonine over serine is merely context-dependent on the specific amino acids that flank the glycosylation site or if there are discrete threonine- and serine-specific isoforms of this transferase. Alternatively, there may be additional factors required to enhance the glycosylation of serine residues in vivo.

Amino Acid Sequence↗
PubMed1993

Functional expression of the rat anion exchanger AE2 in insect cells by a recombinant baculovirus.

We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

A comparison of serine and threonine O-glycosylation by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

O-glycosylated proteins are ubiquitous in eukaryotes and are responsible for a variety of biological functions. O-glycosylation is initiated by the addition of N-acetylgalactosamine to serine or threonine residues, though it is not clear how specific residues are selected for modification. We have compared serine and threonine glycosylation using peptide substrates based on sequences from erythropoietin (EPO) and von Willebrand factor (HVF) that are glycosylated in vivo. UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase was derived from rat parotid, submandibular, and sublingual glands, liver and kidney as well as from human colostrum. The threonine-containing substrates were glycosylated to a much greater extent than those containing serine for all the enzyme sources. Changes in reaction pH, donor concentration, or divalent cation were unable to increase glycosylation of serine. When the incubation time was extended, serine in the EPO-based peptide was found to incorporate GalNAc at a low level, in contrast to the serine-containing HVF peptide, which did not glycosylate at all. By circular dichroism, the non-glycosylating peptide was the only one of the series that did not exhibit random coil structure. Our data suggest that although the structural and sequence requirements for O-glycosylation of serine and threonine residues are similar, serine sites are glycosylated less effectively than are threonine sites in vitro.

Acetylgalactosamine↗