William Kuhns

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 β6-GlcNAc-T) and CMP-sialic acid: Galβ1-3GalNAc-R α3-sialyltransferase (EC 2.4.99.4; α3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 β6-GlcNAc-T activity; core 2 β6-GlcNAc-T from mucin secreting tissue (named core 2 β6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAcβ1-6(GlcNAcβ1-3)GalNAc-R] and blood group I [GlcNAcβ1-6(GlcNAcβ1-3)Galβ-R] branches; core 2 β6-GlcNAc-T in leukemic cells (named core 2 β-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 β6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Galβ1-3GalNAcα-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. α3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Galβ1-3GalNAcα-Bn. Galβ1-3(6-deoxy)GalNAcα-Bn and 3-deoxy-Galβ1-3GalNAcα-Bn competitively inhibited core 2 β6-GlcNAc-T and α3-sialyltransferase activities, respectively.

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Effect of reserpine on the histochemical and biochemical properties of rat intestinal mucin

Biochemical and histochemical parameters of intestinal mucins were examined in control and reserpine-treated rats. An assay for intestinal mucin sulfotransferase was developed and the activity shown to increase 3.4 times over control levels in rats given intraperitonal reserpine (0.5 mg/kg body wt) daily for 7 days. Histochemical staining of intestinal sections revealed an increase in sulfomucins in goblet cells of reserpine-treated rats. The effects were prominent as early as 1 day following injection, particularly in the distal third of the small intestine, and during the next 6 days these changes spread progressively to the middle and proximal thirds. After 3 days of treatment mucins were purified from each intestinal segment and compared to control mucins with respect to composition and [35S]NaSO4 incorporation. Although individual amino acid and carbohydrate molar ratios were unchanged, the total carbohydrate and sulfate content of mucins in treated animals was elevated (two to three times above control) in the middle and distal thirds of the intestine. In vivo [35S]SO4 incorporation into these mucins was also proportionaltely elevated, and was targetted to O-linked oligosaccharide side chains. These findings are consistent with an action of reserpine causing an increased production of mucin which is enriched in glycoprotein components bearing sulfated oligosaccharide chains. The relevance of these findings to the production of hypersulfated and hyperglycosylated mucins in cystic fibrosis is discussed.

Biosynthesis of O-Glycans

The structures and relative amounts of glycan chains found in a glycoprotein are the result of a sensitive regulation of biosynthesis. In the O-linked biosynthetic pathways, sugars are added individually from nucleotide-sugar donors predominantly in the Golgi apparatus by the sequential action of glycosyltransferases (Table 2). The ordered sequence of glycosylation reactions is guided by the relative activity levels and specificities of glycosyltransferases and the intracellular localization of enzymes and substrates. The glycosyltransferases that synthesize N- and O-glycans are apparently arranged in the various compartments of the Golgi as an assembly line where they are placed in the order of their action. With certain exceptions, glycosyltransferases can synthesize only one type of linkage between defined sugar residues.

Structures of α-N-Acetylgalactosamine-Ser/Thr-Linked Oligosaccharides (O-Glycans)

O-glycans have been described on many mammalian and non-mammalian secreted and membrane-bound glycoproteins and may contain GalNAc, Gal, Fuc, G1cNAc and sialic acids, and may be sulfated. Sialic acids may be found as O-acetyl-, N-acetyl-, N-glycolyl- and other derivatives. Mucins comprise the main class of O-glycan and contain glycoproteins which may consist of approximately 50–80% by weight of Oglycans. Mucins are the major glycoprotein components of mucous secretions and also occur on cell membranes.

Cancer of the Urinary and Reproductive Tracts

An association of ovarian cancer with ABO blood groups has been described in large population samples.1 This was confirmed in a recent study of 1261 women with ovarian cancer. Comparison of blood group A patients with a large normal cohort confirmed earlier findings which stated that the cancer risk in the former group was 1.2 with 1 considered as the risk factor in normals.2 A possible explanation is that many tumors which may express Forssman A-like antigen could be eliminated by naturally occurring anti-A in non-A persons; this form of immune surveillance would be absent in A individuals.

Biosynthesis of N-Glycans

The initial steps in the biosynthesis of N-glycans have been are preserved throughout evolution and are similar in lower and higher species. In contrast to O-glycans, N-glycans are pre-assembled as a dolicholpyrophosphate-(Dol-P-P-) intermediate and then transferred to protein by the action of oligosaccharyltransferase in the ER. Subsequent processing includes the trimming of Glc and Man residues and the addition of various sugars by Golgi glycosyltransferases (Fig. 3).

Carbohydrate Deficiency Diseases

Recent refinements in carbohydrate analytical methodology and the application of molecular cloning techniques have defined a whole new area of carbohydrate pathology. Several forms of carbohydrate deficiency diseases have been described. These relate to impairment of glycoprotein processing or biosynthesis.

Glycosylation in Cancer and Oncogenic Transformation

Oncogenic transformation in many tissues is associated with altered glycosylation or the appearance of oncofetal glycoforms (Table 6).1,2 The development of colon cancer occurs in several steps including accumulating activation of oncogenes and inactivation of suppressor genes.3 Glycosylation changes arising during carcinogenesis most likely originate as a consequence of malignant transformation and changing growth and differentiation of the cancer cell. However, these glycosylation changes are critical for the biology of the cancer cell and influence their immunogenicity, cell adhesion and other properties. A mutated gene found in colon cancer appears to code for a member of the adhesion molecule family; this supports the idea that extracellular interactions are important in the control of cell growth.4 The regulation of glycosyltransferase gene expression may be linked to the activation of oncogenes. A major challenge is to understand the control of glycosylation during malignant transformation. As discussed in chapter 8, many factors contribute to the biosynthesis of glycans.

Glycoproteins in Other Cancers

The expression of carbohydrate epitopes can be seen as a process of differentiation in stratified squamous cells. This form of epithelium is observed in the oral cavity, cervix and portions of the esophagus. The progeny of germinal cells in the basal layer changes their topographic position as they differentiate and these cells are eventually exfoliated. Markers such as ABO blood groups tend not to be expressed in basal epithelium but are present in overlying cell layers and are cell cycle-dependent (Table 6).1 Thus, one usually observes a mixed population in stratified epithelium with typical topographic and structural features.