Abstract
A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) Y08564
The reverse transcriptase-PCR product obtained from the human gastric carcinoma cell line MKN45 was cleaved with BstNI, and the remaining uncleaved product was subcloned and sequenced
A BLAST search of the EST data base (GenBank/NCBI) with the coding region of GalNAc-T4 only detected one human GalNAc-T4 EST, which is in contrast to several other GalNAc-transferases that are highly represented by ESTs
Summary
Identification and Cloning of cDNA for GalNAc-T4 —A cDNA sequence (TE4) with extensive similarity to the GalNAc-transferase motif was previously identified following reverse transcriptase-PCR of MKN45 mRNA using degenerate primers [4]. Expression of GalNAc-T4 in Sf9 Cells—Expression constructs designed to contain amino acid residues 32–578 of the coding sequence of the putative GalNAc-T4 gene were prepared by genomic PCR on the two different P1 clones using the primer pair EBHC318 (5Ј-AGCGGATCCTTTTCATGCCTCCGCAGGAGCCG) and EBHC307 with BamHI restriction sites (Fig. 1). These PCR products were cloned into a BamHI site of the expression vector pAcGP67 (Pharmingen), and the expression construct was sequenced. Transferase assays of the full coding expression construct was performed by extracting washed cells in 1% Triton X-100 as described previously [7]. Blots were probed as described previously [4], and washed at 5 ϫ at 42 °C with 2 ϫ SSC, 0.1% SDS, once with 0.5 ϫ SSC, 0.1% SDS, and once at 55 °C with 0.1 ϫ SSC, 0.1% SDS, in a mini-hybridization oven (HYBAID)
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