Cloning and Expression of a Novel, Tissue Specifically Expressed Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family

Hagen, Karl S.; Hagen, Fred K.; Balys, Marlene; Beres, Thomas M.; Wuyckhuyse, B Van; Tabak, Lawrence A.

doi:10.1074/jbc.273.42.27749

Cited by 136 publications

(109 citation statements)

References 23 publications

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“…One example of these is the polypeptide GalNAc-T5 isoform, which is remarkable among Golgi enzymes by having an ϳ500-amino acid-long stem region (2,39). We find that the stem region is extensively O-glycosylated with some 28 O-glycosites detected distributed throughout the stem region.…”

Section: Stshegleedkdhpttstlmentioning

confidence: 82%

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

Vakhrushev

Steentoft

Vester-Christensen

et al. 2013

Molecular & Cellular Proteomics

100

109

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Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of OGalNAc attached to tyrosine. Furthermore, we uncovered ϳ220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes.

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Section: Stshegleedkdhpttstlmentioning

confidence: 82%

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

Vakhrushev

Steentoft

Vester-Christensen

et al. 2013

Molecular & Cellular Proteomics

100

109

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“…S1A). However, GALNTL5 characteristically lacks the typical lectin domain at the C terminus unlike GALNT3, which is also expressed mainly in testis (4,13). The expression of GALNTL5 mRNA was mostly restricted to the human testis (Fig.…”

Section: Resultsmentioning

confidence: 99%

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

Takasaki

Tachibana

Ogasawara

et al. 2014

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

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For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin-proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

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“…Three distinct human GalNAc-transferases, GalNAc-T1, GalNAc-T2, and GalNAc-T3, have been characterised (White et al, 1995;Bennett et al, 1996;Wandall et al, 1997). Recently another 3 homologue enzymes, GalNAc-T4, GalNAc-T5, and GalNAc-T6, have been identified (Bennett et al, 1996(Bennett et al, , 1998(Bennett et al, , 1999Ten Hagen et al, 1998). Compared with the expression of GalNAc-T1 and GalNAc-T2, the expression of GalNAc-T3 is highly tissue specific.…”

mentioning

confidence: 99%

N-acetylgalactosaminyl transferase-3 is a potential new marker for non-small cell lung cancers

et al. 2002

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N-acetylgalactosaminyl transferase-3 (GalNAc-T3) is an enzyme involved in the initial glycosylation of mucin-type O-linked proteins. In the present study, we used immunohistochemistry to examine GalNAc-T3 expression in 215 surgically resected non-small cell lung cancers. We analysed the biological and clinical importance of GalNAc-T3 expression, especially with regard to its potential as a prognostic factor. We found that normal bronchial epithelial cells, bronchial gland cells, and alveolar pneumocytes showed cytoplasmic immunostaining for GalNAc-T3. Low expression of GalNAc-T3, observed in 93 of 215 tumours (43.4%), was found more frequently in tumours from smokers than those from nonsmokers (P=0.001), in squamous cell carcinomas than nonsquamous cell carcinomas (P50.0001), and in moderately and poorly differentiated tumours than well differentiated tumours (P=0.0002). Multivariate logistic regression analysis showed that an association of low GalNAc-T3 expression with squamous cell carcinomas was the only one significant relationship of GalNAc-T3 expression with various factors (P50.0001). Moreover, tumours losing GalNAc-T3 expression had a significantly higher Ki-67 labelling index than tumours retaining GalNAc-T3 expression (P=0.0003). Patients with low GalNAc-T3 expression survived a significantly shorter time than patients with high GalNAc-T3 expression in 103 pStage I non-small cell lung cancers (5-year survival rates, 58% and 78%, respectively; P=0.02 by log-rank test) as well as in 61 pStage I nonsquamous cell carcinomas (5-year survival rates, 63% and 85%, respectively; P=0.03). Low GalNAc-T3 expression was an unfavourable prognostic factor in pStage I non-small cell lung cancers (hazards ratio, 2.04; P=0.03), and in pStage I nonsquamous cell carcinomas (hazards ratio, 2.70; P=0.03). These results suggest that GalNAc-T3 is a new marker of non-small cell lung cancers with specificity for histology and prognosis.

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Cloning and Expression of a Novel, Tissue Specifically Expressed Member of the UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Family

Cited by 136 publications

References 23 publications

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

N-acetylgalactosaminyl transferase-3 is a potential new marker for non-small cell lung cancers

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