Sayuri Hara-Kuge

Intracellular lectins associated with N-linked glycoprotein traffic

The vectorial intracellular transport of N-glycan-linked glycoproteins is indispensable for biological functions. In order to sort these glycoproteins to the correct destination, animal intracellular lectins play important roles as sorting receptors. The roles of such lectins in the biosynthetic pathway from the endoplasmic reticulum (ER) to the cell surface are addressed in this review. Calnexin and calreticulin function via specific carbohydrates in quality control of newly synthesized glycoproteins in the ER, and ERGIC-53 seems to function in the transport of glycoproteins from ER to the Golgi complex. In addition to the well-understood role of mannose 6-phosphate receptor in lysosomal protein sorting, the vesicular integral protein of 36 kDa (VIP36) functions as a sorting receptor by recognizing high-mannose type glycans containing alpha1-->2Man residues for transport from Golgi to the cell surface in polarized epithelial cells.

A Partial Deficiency of Dehydrodolichol Reduction Is a Cause of Carbohydrate-deficient Glycoprotein Syndrome Type I

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is a congenital disorder that involves the underglycosylation of N-glycosylated glycoproteins (Yamashita, K., Ideo, H., Ohkura, T., Fukushima, K., Yuasa, I., Ohno, K., and Takeshita, K. (1993) J. Biol. Chem 268, 5783-5789). In an effort to further elucidate the biochemical basis of CDG syndrome type I in our patients, we investigated the defect in the multi-step pathway for biosynthesis of lipid-linked oligosaccharides (LLO) by the metabolic labeling method using [3H]glucosamine, [3H]mannose, and [3H]mevalonate. The LLO levels in synchronized cultures of fibroblasts from these patients were severalfold lower than those in control fibroblasts in the S phase, and the oligosaccharides released from LLO showed the same structural composition, Glc1∼3·Man9·GlcNAc·GlcNAc, in the case of both the patients and controls. The amount of [3H]mannose incorporated into mannose 6-phosphate, mannose 1-phosphate, and GDP-mannose was greater in fibroblasts from these patients than in the control fibroblasts in the G1 period, although the ratios of these acidic mannose derivatives as indicated by the relative levels of radioactivity were the same for the two types of fibroblasts. Furthermore, upon metabolic labeling with [3H]mevalonate, the level of [3H]dehydrodolichol in fibroblasts from these patients increased in the S phase, and the levels of [3H]dolichol and [3H]dolichol-PP oligosaccharides concomitantly decreased, although the chain length distribution of the respective dolichols and dehydrodolichols was the same in the two types of fibroblasts. These results indicate that the conversion of dehydrodolichol to dolichol is partially defective in our patients and that the resulting loss of dolichol leads directly to underglycosylation.

Identification and characterization of N-acetylglucosamine-6-O-sulfate-specific β1,4-galactosyltransferase in human colorectal mucosa

6‐Sulfo‐sialyl Lewis X structure is attributable to recognition between lymphocytes and high endothelial venules. However, the biosynthetic pathway still remains unclear. We found that a β‐galactosyltransferase (βGalT) in human colorectal mucosa preferentially acts on GlcNAc‐6‐O‐sulfate (6S‐GN). 6S‐GN:β4GalT was partially purified by UDP‐hexanolamine‐Sepharose and asialo‐agalacto‐ovomucin‐Sepharose chromatographies. The optimum pH of this enzyme was found to be 6.5–7.5 and the Michaelis constants for 6S‐GN and UDP‐Gal were 0.43 mM and 16 μM, respectively. The enzymatic activity was dependent on divalent cations and the substrate specificity was not affected by α‐lactalbumin. This is the first demonstration of the occurrence of 6S‐GN:β4GalT.

Localization of VIP36 in the Post-Golgi Secretory Pathway Also of Rat Parotid Acinar Cells

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

Carbohydrate recognition of vesicular integral protein of 36 kDa (ViP36) in intracellular transport of newly synthesized glycoproteins.

Publisher Summary This chapter focuses on the methods to determine the carbohydrate-binding specificity of the vesicular integral protein of 36 kDa (VIP36) to be used in analyzing its roles. VIP36 recognizes high mannose-type glycans and is involved in the transport and sorting of glycoproteins carrying high mannose-type glycans. The methodology for measuring lectin activity of VIP36 discussed includes preparation of recombinant VIP36, preparation of radiolabeled cell lysate, preparation of high mannose-type aspartylglycans (Man7–9GlcNAc2Asn), in vitro binding assay, and preparation of mutant recombinant VIP36. Biological significance of lectin activities of VIP36 in intracellular transport of clusterin is discussed which includes the assay of interaction of VIP36 with clusterin through high mannose-type glycan, plasmid construction and isolation of clones showing stable overproduction, besides thetransport assay of clusterin.