Runx2 Regulates Galnt3 and Fgf23 Expressions and Galnt3 Decelerates Osteoid Mineralization by Stabilizing Fgf23.

Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. Runx2 and Galnt3 were expressed in osteoblasts and osteocytes, and Fgf23 expression was restricted to osteocytes in bone. Overexpression and knock-down of Runx2 upregulated and downregulated, respectively, the expressions of Galnt3 and Fgf23, and Runx2 directly regulated the transcriptional activity of Galnt3 in reporter assays. The expressions of Galnt3 and Fgf23 in osteoblast-specific Runx2 knockout (Runx2fl/flCre) mice were about half those in Runx2fl/fl mice. However, the serum levels of phosphorus and intact Fgf23 in Runx2fl/flCre mice were similar to those in Runx2fl/fl mice. The trabecular bone volume was increased during aging in both male and female Galnt3-/- mice, but the osteoid was reduced. The markers for bone formation and resorption in Galnt3-/- mice were similar to the control in both sexes. Galnt3-/- mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of Galnt3 and Fgf23 and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.

Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase.

The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates (1-4) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.

Oxyresveratrol stimulates mucin production in an NAD+-dependent manner in human intestinal goblet cells.

The intestinal mucus layer plays an important role in the management of inflammatory bowel disease. The aim of this study was to investigate the effects of oxyresveratrol (OXY), an antioxidant, on the stimulation of mucin production in human LS 174T goblet cells and the underlying mechanism thereof. OXY increased MUC2 expression at both the mRNA and protein levels. By performing two-dimensional gel electrophoresis, we found that the expression of nicotinic acid phosphoribosyltransferase1 (NaPRT1) in OXY-treated LS 174T cells was greatly increased compared with that in negative control cells. In addition, the NAD+/NADH ratio was increased in proportion to OXY in LS 174T cells. The expression of NAD+-synthesis enzymes, NaPRT1, nicotinamide riboside kinase1 (NRK1) and nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) was significantly increased at both the mRNA and protein levels in OXY-treated LS 174T cells. The inhibition of NaPRT1 and NRK1 did not decrease MUC2 expression after inhibiting by small interfering RNA (siRNA)-NaPRT1 and siRNA-NRK1, respectively; however, inhibition of Nmnat by an Nmnat inhibitor decreased MUC2 expression in a dose-dependent manner. In conclusion, OXY increases NAD+ levels, resulting in the stimulation of MUC2 expression in LS 174T cells. These findings present a novel role for NAD+ in stimulation of MUC2 expression.

Mapping diet-induced alternative polyadenylation of hypothalamic transcripts in the obese rat.

RNA biogenesis has emerged as a powerful biological event that regulates energy homeostasis. In this context insertion of alternative polyadenylation sites (APSs) dictate the fate of newly synthesized RNA molecules and direct alternative splicing of nascent transcripts. Thus APSs serve a mechanistic function by regulating transcriptome expression and function. In this study we employed a novel RNA-Seq Next Generation Sequencing (NGS) approach that utilized the power of Whole Transcriptome Termini Site Sequencing (WTTS-Seq) to simultaneously measure APS events on multiple RNA biotypes. We used this technique to measure APS events in the hypothalamus of adult male Long Evans rats exposed to a palatable high fat diet (HFD) or chow. Rats maintained on HFD displayed typical hyperphagic feeding and ensuing body weight gain over the one-month manipulation period. Our WTTS-Seq analysis mapped approximately 89,000 unique hypothalamic APSs induced by HFD relative to chow fed controls. HFD exposure produced APSs on multiple RNA biotypes in the hypothalamus. The majority of detected APSs occur on mRNA transcripts that encode functional proteins. Notably we find APSs on micro (miRNA) and long non-coding RNAs (lncRNA), newly recognized transcription factors that regulate body weight in rodents. In addition we detect APSs on protein encoding mRNAs that control neuron projection development and synapse organization and glutamate signaling, key events hypothesized to maintain excess food intake. Importantly, quantitative real time PCR indicated that APS insertion led to increased hypothalamic expression of multiple RNA biotypes. Collectively these data highlight APS events as a novel genetic mechanism that directs hypothalamic RNA biogenesis stimulated by diet-induced obesity.

Effects of sesamin on chondroitin sulfate proteoglycan synthesis induced by interleukin-1beta in human chondrocytes.

BACKGROUND: Numerous studies have reported on the health benefits of sesamin, a major lignin found in sesame (S. indicum) seeds. Recently, sesamin was shown to have the ability to promote chondroitin sulfate proteoglycan synthesis in normal human chondrocytes. This study assesses the anti-inflammatory effect of sesamin on proteoglycans production in 3D chondrocyte cultures. METHODS: To evaluate the effects of sesamin on IL-1ß-treated human articular chondrocytes (HAC) pellets, the pellets were pre-treated with IL-1ß then cultured in the presence of various concentrations of sesamin for 21 days. During that period, the expression of IL-1ß, glycosaminoglycans (GAGs) content and Chondroitin sulfate proteoglycans (CSPGs) synthesis genes (ACAN, XT-1, XT-2, CHSY1 and ChPF) was measured. The GAGs accumulation in the extracellular matrix was determined on day 21 by histological analysis. RESULTS: There was clear evidence that sesamin upregulated expression of all the CSPGs synthesis genes, in contrast to the down-regulation of IL-1ß expression both in genes and in protein levels. The level of release and matrix accumulation of GAGs in IL-1ß pre-treated HAC pellets in the presence of sesamin was recovered. These results correlate with the histological examination which showed that sesamin enhanced matrix CSPGs accumulation. CONCLUSIONS: Sesamin enhances CSPGs synthesis, suppresses IL-1ß expression and ameliorates IL-1ß induced inflammation in human chondrocytes. Sesamin could have therapeutic benefits for treating inflammation in osteoarthritis.

Prognostic role of N-Acetylgalactosaminyltransferase 10 in metastatic renal cell carcinoma.

BACKGROUND AND PURPOSE: A previous study demonstrated that GALNT10 affects the sensitivity of cancer cells to tyrosine kinase inhibitor (TKI) therapy. The aim of this study was to assess whether GALNT10 holds a prognostic role in metastatic renal cell carcinoma (mRCC) patients treated with TKI agents. RESULTS: GALNT10 had no statistical correlation with any other clinicopathological parameters except for route of gaining samples (P = 0.001) and Heng's risk stratification (P = 0.011). Patients with high level of GALNT10 had significantly shorter overall survival (OS) (P < 0.001) and progression-free survival (PFS) (P = 0.002). Importantly, this relationship existed in OS and PFS analyses in sunitinib-treated patients and in OS analyses in sorafenib-treated patients (P = 0.024). In contrast to sorafenib group, percentage of partial response (PR) and stable disease (SD) were higher in sunitinib group, while percentage of progression disease (PD) was much lower. Univariate and multivariate analyses identified that GALNT10 was an independent prognostic factor for OS (HR = 1.938, P = 0.014), not for PFS (HR = 1.532, P = 0.065), in mRCC. Incorporating it into Heng's risk model could sharpen its efficacy in distinguishing patients with potential higher risk. MATERIALS AND METHODS: We retrospectively enrolled 138 mRCC patients treated with sunitinib or sorafenib at Zhongshan Hospital, Shanghai, China. A total of 111 valid cases were finally applied for analyses. CONCLUSIONS: These findings suggest that GALNT10 could be applied as a prognostic marker for OS in mRCC patients.

O-glycosylation regulates polarized secretion by modulating Tango1 stability.

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.

Alterations of membrane lipids and in gene expression of ganglioside metabolism in different brain structures in a mouse model of mucopolysaccharidosis type I (MPS I).

Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform-methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.

A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells.

Mutations in the PHEX gene cause X-linked hypophosphatemia (XLH). Hypophosphatemia in XLH results from increased circulating levels of a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D (calcitriol) synthesis. The current standard therapy for XLH--high-dose phosphate and calcitriol--further increases FGF23 concentrations, suggesting that patients with XLH may have an altered response to extracellular phosphate. To test for the presence of abnormal phosphate responsiveness, we compared serum biochemistries and femoral Fgf23 mRNA expression between wild-type mice, murine models of XLH (Phex(K496X)) and hyperphosphatemic tumoral calcinosis (Galnt3(-/-)), and Galnt3/Phex double-mutant mice. Phex mutant mice had not only increased Fgf23 expression but also reduced proteolytic cleavage of intact Fgf23 protein, resulting in markedly elevated intact Fgf23 levels and consequent hypophosphatemia. In contrast, despite markedly increased Fgf23 expression, Galnt3 knockout mice had significantly high proteolytic cleavage of Fgf23 protein, leading to low intact Fgf23 concentrations and hyperphosphatemia. Galnt3/Phex double-mutant mice had an intermediate biochemical phenotype between wild-type and Phex mutant mice, including slightly elevated intact Fgf23 concentrations with milder hypophosphatemia. Despite the hypophosphatemia, double-mutant mice attempted to reduce serum phosphate back to the level of Phex mutant mice by upregulating Fgf23 expression as much as 24-fold higher than Phex mutant mice. These data suggest that Phex mutations alter the responsiveness of bone cells to extracellular phosphate concentrations and may create a lower set point for "normal" phosphate levels.

Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling.

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.

Small molecule inhibitors of mucin-type O-linked glycosylation from a uridine-based library.

The polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs, also abbreviated ppGaNTases) initiate mucin-type O-linked glycosylation and therefore play pivotal roles in cell-cell communication and protection of tissues. In order to develop new tools for studying mucin-type O-linked glycosylation, we screened a 1338 member uridine-based library to identify small molecule inhibitors of ppGalNAcTs. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. The compounds did not inhibit other mammalian glycosyltransferases or nucleotide sugar utilizing enzymes, suggesting selectivity for the ppGalNAcTs. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. These uridine analogs represent the first generation of chemical tools to study the functions of mucin-type O-linked glycosylation.

Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate.

By a tblastn search with beta 1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in beta 1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5'-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger N-acetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly- and oligosaccharides, and chondroitin sulfate poly- and oligosaccharides with a beta 1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 transcripts were highly expressed in the small intestine, leukocytes, and spleen, however, both CSGalNAcTs were ubiquitously expressed in various tissues.

Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase gene.

Ganglioside functions in tumor metastasis were analyzed by carbohydrate remodeling of a mouse Lewis lung cancer (subline P29) by introducing beta1,4GalNAc-T cDNA. Although P29 was originally a low-metastatic subline in the s.c. injection system, it showed high potential in lung metastasis when i.v.-injected via the tail vein. Two lines of GM(2)(+) transfectants showed markedly reduced metastatic potential to the lung compared to 2 control lines. However, cell proliferation rates and expression levels of various cell adhesion molecules, e.g., integrin family members, SLe(x) and CD44, were essentially unchanged after transfection of the cDNA. Then, cell adhesion to fibronectin-coated dishes was examined, showing that GM(2) (+) transfectants attached to the plates much more slowly than controls, suggesting functional modulation of integrins with newly expressed GM(2). Phosphorylation of the FAK located at downstream of integrin molecules was markedly reduced in GM(2)(+) transfectants, suggesting that GM(2) suppressed cell adhesion signals via fibronectin-integrin interaction.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster.

We report the first demonstration that the activity of a member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family is necessary for viability in Drosophila melanogaster. Expression of the wild-type recombinant pgant35A gene in COS7 cells resulted in in vitro activity against peptide and glycopeptide substrates, demonstrating that this gene encodes a biochemically active transferase. Previous mutagenesis studies identified recessive lethal mutations that were rescued by a genomic fragment containing the pgant35A gene; however, the presence of additional open reading frames within this fragment left open the possibility that another gene was responsible for rescue of the observed lethality. Here, we have determined the molecular nature of the mutations in three independent mutant alleles. Two of the mutant alleles contain premature stop codons within the coding region of pgant35A. The third mutant contains an arginine to tryptophan amino acid change, which, when expressed in COS7 cells, resulted in a dramatic reduction of transferase activity in vitro. PCR amplification of this gene from Drosophila cDNA panels and Northern analysis revealed that it is expressed throughout embryonic, larval, and pupal stages as well as in adult males and females. This study provides the first direct evidence for the involvement of a member of this conserved multigene family in eukaryotic development and viability.

A new UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase mRNA exhibits predominant expression in the hypothalamus, thalamus and amygdala of mouse forebrain.

Protein glycosylation is a common and important process that can alter the stability, half-life, biological activity and receptor recognition of target molecules. We have identified a new putative mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family member, termed GalNAc-T10/ppGaNTase-T10 (gene symbol Galnt10), and determined its expression pattern in mouse CNS using in situ hybridization analysis. Results demonstrated predominant expression of Galnt10 in several distinct hypothalamic, thalamic and amygdaloid nuclei. The most abundant hybridization levels were observed in the paraventricular, ventromedial and arcuate nuclei of the hypothalamus, the anterodorsal and parafascicular nuclei of the thalamus and the central, basomedial and medial nuclei of the amygdala. Expression of Galnt10 was also detected in cerebral cortex, lateral septum, habenula and hippocampus. The localization of this putative glycosyltransferase in distinct regions within the CNS indicates the specificity for complex protein modifications and suggests that region-specific glycosylation represents an essential process in basic biological functions.

The lactose analog GalNAcbeta1-->4Glc is present in bovine colostrum. Enzymatic basis for its occurrence.

We have isolated from bovine colostrum the lactose analog GalNAcbeta1-->4Glc. The enzymatic basis for its occurrence was studied by assaying the activities of GlcNAcbeta-R beta4-N-acetylgalactosaminyltransferase (beta4-GalNAcT) and GlcNAcbeta-R beta4-galactosyltransferase (beta4-GalT) in primary milk and several lactating bovine mammary gland fractions. As the beta4-GalNAcT, which appears to be tightly membrane bound, is induced by the milk protein alpha-lactalbumin (alpha-LA) to act on Glc, it is concluded that beta4-GalNAcT is responsible for the synthesis of GalNAcbeta1-->4Glc in the gland. The comparatively low level (15-20 mg/l) at which this disaccharide is produced may be due to the relatively poor interaction of beta4-GalNAcT with alpha-LA as well as to the fact that alpha-LA does not inhibit the action of the enzyme on N-acetylglucosaminides.

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.

Sequence of bovine carbonic anhydrase VI: potential recognition sites for N-acetylgalactosaminyltransferase.

Carbonic anhydrases (CAs I-VII) are products of a gene family that encodes seven isoenzymes and several CA-related proteins. We report the cloning and sequencing of the cDNA clones encoding one of these isoenzymes, CA VI, from bovine submaxillary gland. The translated polypeptide consists of 319 amino acids, including a signal peptide (14 amino acids) typical of secreted proteins. The predicted mature protein contains 305 amino acids including a 13-amino-acid C-terminal sequence that is also present in the sheep but absent in human CA VI. The deduced mature bovine protein is 87% and 68% identical to that of sheep and human CA VI, respectively. Active-site residues of the enzyme, as well as the three zinc-binding histidines and the two cysteines involved in an intra-chain disulphide bond, are all conserved in the three species. Two potential Asn-glycosylation sites are also conserved, both of which appear to be glycosylated in sheep and bovine CA VI. Two potential peptide recognition sequences are present in bovine CA VI for the glycoprotein hormone: N-acetylgalactosaminyltransferase (GalNAc-transferase), which is one of the two transferases required to form GalNAc-4-SO4 in bovine CA VI-linked oligosaccharides. Specifically, these two sequences are Asp-Leu-Lys-Met-Lys-Lys and Ile-Thr-Lys-Arg-Lys-Lys. Comparison of these sequences with sheep and human CA VI sequences indicates that distinct glycoforms of CA VI could exist in submaxillary gland from different species.

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.

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.