Lawrence A. Tabak

Control of mucin-type O-glycosylation: A classification of the polypeptide GalNAc-transferase gene family

Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.

Structural Aspects of Salivary Glycoproteins

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MGI ) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO+GLY+GLN. PRGs secondary structure is comprised of 70% random coil (naked regions) and 30% β-turns (glycosylated domains). MGI (>103 kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAcα2, 3Galβ1, 3GalNAc, Galβ1, 3GalNAc, and Fucα1, 2Galβ1, 3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MGI and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part, on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

Handbook of glycosyltransferases and related genes

The CHST14 gene, localized at 15q14, is a single exon gene with an open reading frame of 1131 base pairs, encoding a 43 kDa protein dermatan-4-Osulfotransferase-1 (D4ST1) that catalyzes the 4-O-sulfation of N-acetyl-D-galactosamine residues in dermatan sulfate (DS). Both nearly exhaustively desulfated DS and partially desulfated DS serve as excellent substrates for the enzyme. Chst14/D4st1-deficient mice showed growth retardation as well asmultiple system abnormalities including neurology such as decreased neurogenesis and diminished T. Kosho (*) School of Medicine, Department of Medical Genetics, Shinshu University, Matsumoto, Japan e-mail: ktomoki@shinshu-u.ac.jp S. Mizumoto • K. Sugahara Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Kita-ku, Sapporo, Japan e-mail: mizumoto@sci.hokudai.ac.jp; k-sugar@sci.hokudai.ac.jp N. Taniguchi et al. (eds.), Handbook of Glycosyltransferases and Related Genes, DOI 10.1007/978-4-431-54240-7_156, # Springer Japan 2014 1135 proliferation of neural stem cells. Recently, recessive loss-of-function mutations in the CHST14 gene were found to cause a specific form of Ehlers-Danlos syndrome (EDS) designated as D4ST1-deficient EDS (DD-EDS). The disorder is characterized by progressive multisystem fragility-related manifestations (skin hyperextensibilty and fragility, progressive spinal and foot deformities, large subcutaneous hematoma) and various malformations (facial features, congenital eye/heart/gastrointestinal defects, congenital multiple contractures). Glycosaminoglycan (GAG) chains from the affected skin fibroblasts were composed of a negligible amount of DS and excess chondroitin sulfate (CS), which was suggested to result from an impaired lock by 4-O-sulfation due to D4ST1 deficiency followed by back epimerization from L-iduronic acid to D-glucuronic acid. GAG chains of decorin from the affected skin fibroblasts were composed exclusively of CS and no DS, the opposite features observed in normal controls. Thus, skin fragility in the disorder was supposed to be caused by impaired assembly of collagen fibrils mediated by decorin bearing a CS chain that replaced a DS chain. The disorder stresses the importance of the role of CHST14/ D4ST1 and DS in human development and maintenance of extracellular matrices.

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

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptideN-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controllingO-linked glycosylation.

Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcussanguis with specificity for a NeuAcα2,3Ga1β1,3Ga1NAc sequence

Abstract Evidence is presented for the presence of a lectin on Streptococcus sanguis with specificity towards the major acidic oligosaccharide of human salivary mucin. Based upon hemagglutination inhibition studies, the strongest inhibitor was NeuAcα2,3Galβ1,3GalNAcol ⪢ NeuAcα2,3Galβ1,4Glc ⪢ NeuAc > Gal. Interactions were not heat sensitive or charge dependent, and were not affected by the presence of bacterial cell associated neuraminidase. The lectin could be extracted from Streptococcus sanguis with lithium 3,5-diiodosalicylate (LIS). Incubation of LIS extracts with carbohydrate ligands demonstrated that the specificity of binding was NeuAcα2,3Galβ1,3[ 3 H-]GalNAcol ⪢ Galβ1,3[ 3 H-]GalNAcol .

Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva.

A low-molecular-weight, monomeric, mucin-type glycoprotein (MG2) has been isolated from human submandibular-sublingual saliva. Initial purification involved sequential gel-filtration on Sephadex G-200 and Sepharose CL-2B, the latter in the presence of 6M urea. Fractions containing MG2 were next separated from contaminating secretory IgA by immunoaffinity chromatography or recycling through Sephadex G-200. Mucin fractions were 14C-labeled by reductive methylation, and then the final purification-step entailed recycling radiolabeled materials through Sephadex G-200. Radiolabeling aided in the assessment of purity, as judged by SDS-PAGE and ion-exchange chromatography. The carbohydrate portion accounted for 69.6% of the recovered weight and was composed of N-acetyl-glucosamine, N-acetylgalactosamine, galactose, fucose, and N-acetylneuraminic acid. Sulfate was also present. The protein comprised 30.4% of the recovered weight with threonine, serine, proline, and glycine accounting for 75.2% of the total amino acids. The oligosaccharides were alkali-labile, indicating an O-glycosyl linkage to the peptide. The mucin was weakly acidic and had an estimated mol. wt. of 200 000-250 000.

Dynamic Association between the Catalytic and Lectin Domains of Human UDP-GalNAc:Polypeptide {alpha}-N-Acetylgalactosaminyltransferase-2

The family of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs) is unique among glycosyltransferases, containing both catalytic and lectin domains that we have previously shown to be closely associated. Here we describe the x-ray crystal structures of human ppGalNAcT-2 (hT2) bound to the product UDP at 2.75 Å resolution and to UDP and an acceptor peptide substrate EA2 (PTTDSTTPAPTTK) at 1.64 Å resolution. The conformations of both UDP and residues Arg362–Ser372 vary greatly between the two structures. In the hT2-UDP-EA2 complex, residues Arg362–Ser373 comprise a loop that forms a lid over UDP, sealing it in the active site, whereas in the hT2-UDP complex this loop is folded back, exposing UDP to bulk solvent. EA2 binds in a shallow groove with threonine 7 positioned consistent with in vitro data showing it to be the preferred site of glycosylation. The relative orientations of the hT2 catalytic and lectin domains differ dramatically from that of murine ppGalNAcT-1 and also vary considerably between the two hT2 complexes. Indeed, in the hT2-UDP-EA2 complex essentially no contact is made between the catalytic and lectin domains except for the peptide bridge between them. Thus, the hT2 structures reveal an unexpected flexibility between the catalytic and lectin domains and suggest a new mechanism used by hT2 to capture glycosylated substrates. Kinetic analysis of hT2 lacking the lectin domain confirmed the importance of this domain in acting on glycopeptide but not peptide substrates. The structure of the hT2-UDP-EA2 complex also resolves long standing questions regarding ppGalNAcT acceptor substrate specificity.

Reduced chemotactic peptide binding in juvenile periodontitis: A model for neutrophil function

Abstract Chemotactic peptide binding sites from peripheral blood neutrophils of Localized Juvenile Periodontitis patients and normal controls were quantitated using tritiated N-formylmethionylleucyl-phenylalanine and a rapid filtration assay. It was found that there is a significant reduction in the number of binding sites per cell on neutrophils from the patient group whereas binding affinity remained the same as control values. A direct correlation between in vitro neutrophil chemotactic response and density of binding sites was found. Since these patients present little clinical illness, localized juvenile periodontitis can be a valuable model for the study of human neutrophil function.

CHARACTERIZATION OF A UDP-GALNAC:POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE THAT DISPLAYS GLYCOPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE ACTIVITY

We report the cloning, expression, and characterization of a novel member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family that transfers GalNAc to a GalNAc-containing glycopeptide. Northern blot analysis revealed that the gene encoding this enzyme, termed ppGaNTase-T6, is expressed in a highly tissue-specific manner. Significant levels of transcript were found in rat and mouse sublingual gland, stomach, small intestine, and colon; trace amounts were seen in the ovary, cervix, and uterus. Recombinant constructs were expressed transiently in COS7 cells but demonstrated no transferase activity in vitro against a panel of unmodified peptides, including GTTPSPVPTTSTTSAP (MUC5AC). However, when incubated with the total glycosylated products obtained by action of ppGaNTase-T1 on MUC5AC (mainly GTT(GalNAc)PSPVPTTSTT(GalNAc)SAP), additional incorporation of GalNAc was achieved, resulting in new hydroxyamino acids being modified. The MUC5AC glycopeptide failed to serve as a substrate for ppGaNTase-T6 after modification of the GalNAc residues by periodate oxidation and sodium borohydride reduction, indicating a requirement for the presence of intact GalNAc. This suggests thatO-glycosylation of multisite substrates may proceed in a specific hierarchical manner and underscores the potential complexity of the processes that regulate O-glycosylation.

Artificial Salivas: Present and Future:

Modern technology has allowed us to understand better the functions of saliva and now provides a rationale for developing: (1) diagnostic reagents for monitoring oral and systemic health status and (2) replacement therapies for individuals with salivary dysfunctions. Several areas of dental research are directed at augmenting or enhancing both the quality and quantity of saliva for individuals with dry mouth. An “intrinsic” approach is being explored which utilizes medications such as pilocarpine and bromhexine to stimulate the salivary glands to produce more saliva. An “extrinsic” approach proposes to use topically applied artificial saliva. Studies in our laboratory have been directed toward developing artificial salivas which incorporate many of the protective features of “native” saliva. An ideal artificial saliva should be “long-lasting”, provide lubrication, inhibit colonization of microflora responsible for dental caries and gingivitis, and coat the oral soft tissues for protection against environmental insult and desiccation. Studies are currently under way to determine the structural requirements of salivary molecules responsible for these protective functions. Composite salivary molecules consisting of multiple biologically active or “functional domains” could then be designed and synthesized based upon primary sequence and conformational analyses, computer-assisted structural predictions, and in vitro testing. These supersalivary substances could then be used as saliva substitutes for targeting to selected oral surfaces to promote mineralization, hydration, and/or regulate microbial-mediated disease.