Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene

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Abstract

O-Linked N-acetylglucosamine (GlcNAc) transferase (OGT) mediates a novel hexosamine-dependent signal transduction pathway. Yet, little is known about the regulation of ogt gene expression in mammals. We report the sequence and characterization of the mouse ogt locus and provide a comparison with the human and rat ogt genes. The mammalian ogt genes are similar in structure and exhibit ∼80% sequence identity. The mouse and human ogt genes contain two potential promoters producing four major transcripts. By analyzing 56 human cDNA clones and other existing expressed sequence tags, we found that at least three protein products differing at their amino terminus result from alternative splicing. We used OGT-specific antisera to demonstrate the presence of these isoforms in HeLa cells. The longest form is a nucleocytoplasmic OGT (ncOGT) with 12 tetratricopeptide repeats (TPRs); a shorter form of OGT encodes a mitochondrially sequestered enzyme with 9 TPRs and an N-terminal mitochondrion-targeting sequence (mOGT). An even shorter form of OGT (sOGT) contains only 2 TPRs. The genomic organization of OGT appears to be highly conserved throughout metazoan evolution. These results provide the basis for a more detailed analysis of the significance and regulation of the nucleocytoplasmic and mitochondrial isoforms of OGT in mammals.

Section snippets

Mouse tissue distribution of OGT

The Mouse Multiple Choice Northern blots were purchased from OriGene Technologies (Rockville, MD). The blots were prehybridized in 1% bovine serum albumin, 0.5 M NaPO4, pH 7.0, 1 mM EDTA, 7% sodium dodecyl sulfate, and 100 μg/ml denatured salmon testis DNA at 55 °C for 2 h and then hybridized overnight at 55 °C with the gel-purified [α-32P]ATP-radiolabeled human ogt EcoRV–XbaI fragment and the mouse OGT PCR probe (350 bp). The blots were washed twice for 15 min with 0.5% bovine serum albumin, 5% sodium

Tissue distribution of mouse OGT

To examine the relative size and abundance of ogt transcripts in various mouse tissues, Northern blot analysis was performed (Fig. 1). The human ogt probe identified several distinct bands between 9.5 and 4 kb which are present in different amounts in the various mouse tissues tested. Heart and muscle exhibited a relative enrichment of the 6.5 kb species. The exception was stomach, which contained very little of the ogt transcripts. A mouse ogt PCR probe corresponding to the 5 end of a mouse

Mammalian ogt is highly conserved and is predicted to contain two promoters

The genes encoding the human and rodent OGT are highly conserved in both overall structure and sequence. Several promoter prediction algorithms we employed suggest that ogt may contain two promoters. The most upstream of these contains a prototypic CpG island motif and likely represents the “housekeeping” promoter for OGT. The sequence 1000 bp upstream of the presumptive transcription start contains a large number of transcription factor binding sites. The putative downstream promoter would be

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