Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides.

The recombinant proteins of the two novel UDP-N-acetylgalactosamine (GalNAc) glycopeptide:N-acetylgalactosaminyltransferases (designated gpGaNTase-T7 and gpGaNTase-T9) were assayed with O-glycosylated products obtained from the prior action of the ubiquitous transferases (GaNTase-T1 and GaNTase-T2) towards MUC5AC mucin motif peptides (GTTPSPVPTTSTTSAP and peptides with single amino acid substitutions, GTTPSAVPTTSTTSVP and GTTPSPVPTTSITSVP, that are a reflection of mucin molecule polymorphism). gpGaNTase-T9 is known to be expressed differentially and more abundantly than gpGaNTase-T7 in some tissues; the results of in vitro glycosylation also indicates a difference in acceptor substrate specificities between the gpGaNTase isoforms. With the use of capillary electrophoresis, MS and Edman degradation, our study suggests that, in the O-glycosylation of mucin-type proteins, approach and recognition signalling by gpGaNTase-T7 and gpGaNTase-T9 depend largely on the peptide's primary structure (for example the presence of multiple clusters of hydroxy amino acids and the number of GalNAc residues attached to the peptide backbone). O-glycosylation in terms of sites of attachment seems to be less random than previously described and, if sequential reactions are ordered throughout the Golgi stack, the complete O-glycosylation of the mucin molecules seems to be finely tuned to respond to specific damage to, or attack on, epithelia.

Targeted disruption of the Nhe1 gene fails to inhibit beta(1)-adrenergic receptor-induced parotid gland hypertrophy.

Chronic beta(1)-adrenergic receptor activation results in hypertrophy and hyperplasia of rodent salivary gland acinar cells. Na(+)/H(+) exchanger isoform 1 (NHE1) regulates cell volume and the induction of cell proliferation in many tissues. To investigate the relationship between NHE1 and the response of parotid glands to beta(1)-adrenergic agonists, we examined by Northern blot analysis NHE1 expression in saline-treated mice and mice 30 min and 2, 6, and 24 h after isoproterenol injection. NHE1 transcripts increased approximately 50% by 2 h, and a more than twofold increase was noted at 24 h. Isoproterenol did not acutely increase Na(+)/H(+) exchanger activity; however, exchanger activity was significantly elevated by 24 h. To test whether NHE1 activity is essential for inducing salivary gland hypertrophy in vivo, mice with targeted disruption of Nhe1 were treated with isoproterenol. Na(+)/H(+) exchanger activity was absent in acinar cells from Nhe1(-/-) mice, nevertheless, the lack of NHE1 failed to inhibit isoproterenol-induced hypertrophy. These data directly demonstrate that acinar cell hypertrophy induced by chronic beta(1)-adrenergic receptor stimulation occurs independently of NHE1 activity.

Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9.

We have cloned, expressed and characterized the gene encoding a ninth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein consists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydrophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encoding this enzyme is expressed in a broadly distributed manner across many adult tissues. Significant levels of 5- and 4.2-kilobase transcripts were found in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed significant hybridization in the developing mandible, maxilla, intestine, and mesencephalic ventricle. Constructs expressing this gene transiently in COS7 cells resulted in no detectable transferase activity in vitro against a panel of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and EA2 (PTTDSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obtained by the prior action of ppGaNTase-T1), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acid modification. The activity of this glycopeptide transferase is distinguished from that of ppGaNTase-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-glycopeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species. This isoform thus represents the second example of a glycopeptide transferase and is distinct from the previously identified form in enzymatic activity as well as expression in embryonic and adult tissues. These findings lend further support to the existence of a hierarchical network of differential enzymatic activity within the diversely regulated ppGaNTase family, which may play a role in the various processes governing development.

Low levels of GRP-Ca expression in transgenic mice.

The GRP-Ca gene of the rat encodes a member of the glutamine/glutamic-acid-rich protein (GRP) family. This gene is expressed in a highly tissue-specific fashion, with transcription being found only in the acinar cells of the submandibular gland (SMG). To begin to define the cis-acting elements governing GRP-Ca expression, we constructed transgenic mice containing the rat GRP-Ca gene plus 9.5 kb of 5' genomic sequence and 1 kb of 3 genomic sequence. Expression of GRP-Ca was detectable in progeny from only 1 of 3 independent founders. Expression levels of the transgenic GRP-Ca were much lower than the native GRP-Ca found in the rat SMG. Furthermore, GRP-Ca in transgenic mice was not tissue-specifically expressed, being found in both the SMG/SLG complex and the liver. These results indicate that the genomic region of GRP-Ca included in these transgenic mice is not sufficient to confer the high levels of tissue-specific expression seen in the rat.

Characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity.

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests that O-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family.

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controlling O-linked glycosylation.

Chromosomal organization and expression analysis of two distinct genes encoding glutamine/glutamic acid-rich proteins.

GRP-Ca and GRP-Cb are two genes that encode glutamine/glutamic acid-rich proteins of the rat. These genes are very similar in structure and sequence, differing only within an approx. 90 bp segment of exon 3. We have used distinct oligonucleotide probes to unambiguously distinguish GRP-Ca and GRP-Cb gene expression. The two genes are expressed to relatively equivalent levels only in the submandibular gland. Chronic daily exposure to the beta-adrenergic agonist, isoprenaline, resulted in a statistically significant decrease in GRP-Ca expression, with no effect on GRP-Cb, in contrast with previous reports. Furthermore it was determined by PCR analysis of both submandibular-gland cDNA and genomic DNA that the GRP-Cb gene shows interanimal variability in the number of 69 bp tandem repeats found within exon 3; GRP-Cb genes were shown to contain four, five or six repeats. GRP-Ca shows no such variability, containing only five tandem repeats in all animals examined. The two genes were localized to within 450 kb of one another at q43-q44 of rat chromosome 4 using somatic cell hybrid analysis, pulsed-field gel analysis and fluorescent in situ hybridization.

cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.

The cDNA for a fourth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, termed ppGaNTase-T4, has been cloned from a murine spleen cDNA library and expressed transiently in COS7 cells as a secreted functional enzyme. Degenerate primers, based upon regions that are conserved among the known mammalian members of the enzyme family (ppGaNTase-T1, -T2, and -T3) and three Caenorhabditis elegans homologues (ppGaNTase-TA, -TB, and -TC), were used in polymerase chain reactions to identify and clone this new isoform. Substrate preferences for recombinant murine ppGaNTase-T1 and ppGaNTase-T4 isozymes were readily distinguished. ppGaNTase-T1 glycosylated a broader range of synthetic peptide substrates; in contrast, the ppGaNTase-T4 preferentially glycosylated a single substrate among the panel of 11 peptides tested. Using Northern blot analysis, a ppGaNTase-T4 message of 5.5 kilobases was detectable in murine embryonic tissues, as well as the adult sublingual gland, stomach, colon, small intestine, lung, cervix, and uterus with lower levels detected in kidney, liver, heart, brain, spleen, and ovary. Thus, the pattern of expression for ppGaNTase-T4 is more restricted than for the three previously reported isoforms of the enzyme. The variation in expression patterns and substrate specificities of the ppGaNTase enzyme family suggests that differential expression of these isoenzymes may be responsible for the cell-specific repertoire of mucin-type oligosaccharides on cell-surface and secreted O-linked glycoproteins.

Charge distribution of flanking amino acids inhibits O-glycosylation of several single-site acceptors in vivo.

From surveys of known O-glycosylation sites and in vitro glycosylation assays with synthetic peptide acceptors, it appears that the presence of charged amino acids near serine/threonine residues reduces the likelihood of O-glycosylation by UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferases (ppGaNTases). Previously, we demonstrated that the in vivo O-glycosylation of a sequence derived from a known glycosylation site of human von Willebrand factor (PHMAQVTVGPGL) was markedly reduced when charged residues were substituted at position -1 and +3 relative to the single threonine. In contrast, acidic residues at positions -2, +1, and +2 had no effect (Nehrke et al., 1996), suggesting that charge distribution but not charge density was important. To determine whether the charge distribution effect on O-glycosylation is limited to a specific sequence context or restricted to unique isoforms of ppGaNTase, we have analyzed the in vivo O-glycosylation of six secreted recombinant reporter proteins in three different cell backgrounds. The differential presence of known ppGaNTase transcripts was determined in each cell type by Northern blot analysis. Each reporter, which contains a single site of O-glycosylation, was O-glycosylated in a cell-background-specific manner; digestion with O-glycanase and alpha-N-acetylgalactosaminidase following mild acid hydrolysis suggested that simple type II core structures were acquired. However, in COS7 cells, one reporter peptide acquired glycosaminoglycans in preference to mucin-type O-glycans. Regardless of cell background or the reporter examined, the substitution of glutamic acid residues at positions -1 and +3 markedly diminished the level of mucin-type O-glycosylation. Charge distribution would appear, therefore, to play a more general role in determining the extent to which solitary O-glycosylation sites are modified.

Cloning and expression of mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T3.

A novel isoform of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated ppGaNTase-T3, has been cloned from a mouse testis cDNA library and expressed in COS7 cells. ppGaNTase-T3 displayed 64 and 59% amino acid identity with ppGaNTase-T1 and ppGaNTase-T2, respectively, and 96% amino acid identity with the recently reported human form of ppGaNTase-T3. The ppGaNTase-T3 transcript is abundant in the major salivary glands, gastrointestinal tract and both the male and female reproductive systems. ppGaNTase-T3 and ppGaNTase-T1 display overlapping substrate preferences in vitro, although mapping studies of O-glycosylated peptides suggests that certain hydroxyamino acids are preferentially glycosylated by each isoform. This suggests that more than one isoform of ppGaNTase may be required to complete the O-glycosylation of endogenous substrates.

Disparate replication properties of integrated and extrachromosomal forms of bovine papilloma virus in ID13 cells.

Bovine papillomavirus (BPV) previously has been reported to exist in transformed rodent cell lines as both chromosomally integrated and extrachromosomal forms. In the BPV-transformed mouse cell line ID13, extrachromosomal BPV molecules replicate throughout S phase of the cell cycle in a random choice mode. We report here that these replication properties were altered for chromosomally integrated BPV DNA in five independent ID13 subclones. In all of the subclones, the integrated BPV sequences, which had no detectable deletions or mutations, existed in head-to-tail tandem arrays that replicated once per cell cycle, predominantly late in S phase. In contrast, extrachromosomal BPV molecules present in other subclones of the same cell line replicated in the random choice mode observed previously for non-integrated BPV. Our results indicate that the replication origin of integrated BPV either is inactivated as a consequence of chromosomal insertion, leading to the replication of BPV from origins in the flanking chromosomal DNA, or alternatively is reprogrammed to function in a once-per-cell cycle mode predominantly late in S phase.

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.

Timing of replication of beta satellite repeats of human chromosomes.

The beta satellite sequences of the human genome are a family of genetic elements consisting of 68-69 bp monomeric units repeated contiguously in long arrays up to 1 Mb in length. We have determined the timing of replication of beta satellite subgroups located in the heterochromatic portion of chromosome 9 and on the acrocentric chromosomes in regions both distal and proximal to the rDNA genes. We report that these dispersed subgroups of beta satellite sequences all replicate late during S phase of the cell cycle.

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.