Helle Hassan

CDNA CLONING AND EXPRESSION OF A NOVEL HUMAN UDP-N-ACETYL-ALPHA -D-GALACTOSAMINE : POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE, GALNAC-T3

The glycosylation of serine and threonine residues during mucin-type O-linked protein glycosylation is carried out by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferase). Previously two members, GalNAc-T1 and −T2, have been isolated and the genes cloned and characterized. Here we report the cDNA cloning and expression of a novel GalNAc-transferase termed GalNAc-T3. The gene was isolated and cloned based on the identification of a GalNAc-transferase motif (61 amino acids) that is shared between GalNAc-T1 and −T2 as well as a homologous Caenorhabditis elegans gene. The cDNA sequence has a 633-amino acid coding region indicating a protein of 72.5 kDa with a type II domain structure. The overall amino acid sequence similarity with GalNAc-T1 and −T2 is approximately 45%; 12 cysteine residues that are shared between GalNAc-T1 and −T2 are also found in GalNAc-T3. GalNAc-T3 was expressed as a soluble protein without the hydrophobic transmembrane domain in insect cells using a Baculo-virus vector, and the expressed GalNAc-transferase activity showed substrate specificity different from that previously reported for GalNAc-T1 and −T2. Northern analysis of human organs revealed a very restricted expression pattern of GalNAc-T3.

Cloning of a Human UDP-N-Acetyl-α-d-Galactosamine:PolypeptideN-Acetylgalactosaminyltransferase That Complements Other GalNAc-Transferases in Complete O-Glycosylation of the MUC1 Tandem Repeat

A fourth human UDP-GalNAc:polypeptideN-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.

Cloning and Characterization of a Close Homologue of Human UDP-N-acetyl-α-d-galactosamine:Polypeptide N-Acetylgalactosaminyltransferase-T3, Designated GalNAc-T6 EVIDENCE FOR GENETIC BUT NOT FUNCTIONAL REDUNDANCY

The UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.

A Family of Human β3-Galactosyltransferases CHARACTERIZATION OF FOUR MEMBERS OF A UDP-GALACTOSE:β-N-ACETYL-GLUCOSAMINE/β-NACETYL-GALACTOSAMINE β-1,3-GALACTOSYLTRANSFERASE FAMILY

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase, designated β3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to β3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyltransferase (β3Gal-T2) with similar kinetic properties as β3Gal-T1. Another gene represented a UDP-galactose:β-N-acetyl-galactosamine β-1,3-galactosyltransferase (β3Gal-T4) involved in GM1/GD1 ganglioside synthesis, and this gene was highly similar to a recently reported rat GD1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794–24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. β3Gal-T1 mRNA was expressed in brain, β3Gal-T2 was expressed in brain and heart, and β3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. β3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous β3-galactosyltransferase genes.

A NOVEL HUMAN UDP-N-ACETYL-D-GALACTOSAMINE:POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE, GALNAC-T7, WITH SPECIFICITY FOR PARTIAL GALNAC-GLYCOSYLATED ACCEPTOR SUBSTRATES

A novel member of the human UDP‐N‐acetyl‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase gene family, designated GalNAc‐T7, was cloned and expressed. GalNAc‐T7 exhibited different properties compared to other characterized members of this gene family, in showing apparent exclusive specificity for partially GalNAc‐glycosylated acceptor substrates. GalNAc‐T7 showed no activity with a large panel of non‐glycosylated peptides, but was selectively activated by partial GalNAc glycosylation of peptide substrates derived from the tandem repeats of human MUC2 and rat submaxillary gland mucin. The function of GalNAc‐T7 is suggested to be as a follow‐up enzyme in the initiation step of O‐glycosylation.

Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions.

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitroaddition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcα or Galβ1–3GalNAcα at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position −6 (Thr-10) or −10 (Ser-6) and is inhibited by disaccharide at position −11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.

A lectin recognizes differential arrangements of O-glycans on mucin repeats

Interaction of Vicia villosa agglutinin-B4 (VVA-B4) to glycopeptides with O-linked GalNAc residues was investigated by surface plasmon resonance. The affinity was shown to be influenced by the arrangement of O-glycosylation sites on a peptide, PTTTPITTTTK, representing the tandem repeat of MUC2. The association rate constant was relatively high with a particular category of GalNAc-peptides in which more than three amino acid residues were placed between GalNAc-Thr residues. PTT( *)T( *)PITT( *)T( *)TK (T( *) indicates GalNAc-Thr) had the highest association rate constant among the glycopeptides tested. The dissociation rate constant was low in the peptides containing consecutive GalNAc residues and PT( *)TTPIT( *)T( *)T( *)TK was the lowest of the glycopeptides tested. Dissociation constant (K(D)), calculated as k(d)/k(a) was the lowest with PTT( *)T( *)PITT( *)T( *)TK. Therefore, the arrangement but not the quantity of GalNAc residues apparently determines the affinity between VVA-B4 and peptides with attached GalNAc residues.

Incorporation of N-acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N-acetyl-D-galactosamine transferase-T1, T2 and T3.

An oligopeptide containing three consecutive Thr residues mimicking the tandem repeat portion of MUC2 (PTTTPLK) was investigated for the acceptor specificity to UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase isozymes, UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, T2 and T3. The enzymatic reaction products were fractionated by the reversed‐phase high performance liquid chromatography, then characterized by matrix‐assisted laser desorption ionization time of flight mass spectrometry and by a peptide sequencing analysis. A maximum of two, one or three N‐acetyl‐D‐galactosamine residues was transferred by UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, T2 or T3, respectively. The preferential orders of N‐acetyl‐D‐galactosamine incorporation were Thr‐2, then Thr‐4 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, Thr‐2 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T2 and Thr‐4, Thr‐3, then Thr‐2 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T3.

Characterization of a Panel of Monoclonal Antibodies Using GalNAc Glycosylated Peptides and Recombinant MUC1

A panel of 56 murine monoclonal antibodies submitted to the ISOBM TD-4 (MUC1) Workshop was analysed for reactivity against nonglycosylated and in vitro GalNAc glycosylated peptides. Twenty-six antibodies reacted with nonglycosylated MUC1 peptides containing 3–5 tandem repeats. The reactivities of most of the antibodies were not affected by GalNAc glycosylation of the peptides. Antibody #147 (decoded as BCP9) reactivity was inhibited when 3 mol of GalNAc per repeat were incorporated in the peptide (at sites T in GVTSA and ST in GSTAP), whereas the reactivity with GalNAc glycosylated peptides with 2 mol of GalNAc per repeat (sites T in GVTSA and T in GSTAP) was unaffected. This is in agreement with the epitope defined as GSTAP.