We report the cloning and partial characterization of the fourth member of the vertebrate heparan sulfate/ heparin: GlcNAc N-deacetylase/GlcN N-sulfotransferase family, which we designate NDST4. Full-length cDNA clones containing the entire coding region of 872 amino acids were obtained from human and mouse cDNA libraries. The deduced amino acid sequence of NDST4 showed high sequence identity to NDST1, NDST2, and NDST3 in both species. NDST4 maps to human chromosome 4q25-26, very close to NDST3, located at 4q26 -27. These observations, taken together with phylogenetic data, suggest that the four NDSTs evolved from a common ancestral gene, which diverged to give rise to two subtypes, NDST3/4 and NDST1/2. Reverse transcriptionpolymerase chain reaction analysis of various mouse tissues revealed a restricted pattern of NDST4 mRNA expression when compared with NDST1 and NDST2, which are abundantly and ubiquitously expressed. Comparison of the enzymatic properties of the four murine NDSTs revealed striking differences in N-deacetylation and N-sulfation activities; NDST4 had weak deacetylase activity but high sulfotransferase, whereas NDST3 had the opposite properties. Molecular modeling of the sulfotransferase domains of the murine and human NDSTs showed varying surface charge distributions within the substrate binding cleft, suggesting that the differences in activity may reflect preferences for different substrates. An iterative model of heparan sulfate biosynthesis is suggested in which some NDST isozymes initiate the N-deacetylation and N-sulfation of the chains, whereas others bind to previously modified segments to fill in or extend the section of modified residues.Heparan sulfate and heparin bind a variety of growth factors, enzymes, and extracellular matrix proteins (1). These interactions depend on specific arrangements of variably sulfated glucosaminyl residues (GlcNAc, GlcN, 1 and GlcNS) and glucuronic (GlcA) and iduronic acids. The assembly of these sequences proceeds in a stepwise manner as follows. (i) The chains initiate by formation of the linkage tetrasaccharide, GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤, on serine residues of core proteins; (ii) the chains elongate by alternating the additions of GlcNAc␣1,4 and GlcA␤1,4 residues; (iii) the chains are modified initially by N-deacetylation and N-sulfation of subsets of GlcNAc residues, (iv) adjacent D-GlcA residues undergo C5-epimerization to L-iduronic acid, and (v) sulfation occurs at C2 of the uronic acid residues and at C6 and C3 of glucosaminyl residues. In this scheme, GlcNAc N-deacetylation and N-sulfation creates the prerequisite substrate needed for the later modification reactions (reviewed by Rodén (2)). These reactions are catalyzed by a family of enzymes designated the GlcNAc N-deacetylase/N-sulfotransferases (NDSTs).Three NDST isozymes have been identified in vertebrates, whereas only single orthologs are known in Drosophila melanogaster and Caenorhabditis elegans (3-11). Mutations in these genes can have profound effects on development. In D. melanogas...

The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase͞N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker ␣-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-Osulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.glycosaminoglycans ͉ sulfation ͉ epimerization ͉ enzyme localization ͉ Golgi complex T he biosynthesis of heparan sulfate initiates by the translation of a proteoglycan core protein and the assembly of the so-called linkage region tetrasaccharide on specific serine residues (-GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-O-Ser). The chain then polymerizes by the addition of alternating N-acetylglucosamine (GlcNAc␣1,4) and glucuronic acid (GlcA␤1,4) residues. A series of modification reactions takes place simultaneously that involves at least six enzymatic activities: (i) N-deacetylation of a portion of GlcNAc residues, (ii) N-sulfation of the resulting unsubstituted amino groups to form GlcNS units, (iii) 5-epimerization of adjacent D-GlcA residues to form L-iduronic acid (IdoA), (iv) 2-O-sulfation of L-IdoA and more rarely D-GlcA residues, (v) 6-O-sulfation of glucosamine units, and (vi) occasional 3-O-sulfation of glucosamine residues (Fig. 1A). A major question concerns how these enzymes orchestrate the formation of specific oligosaccharide sequences with unique binding properties for ligands (1, 2). Multiple isozymes exist for several of the transferases that differ in substrate specificity and temporal͞spatial expression during developmen...

N-Deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin initiate a series of chemical modifications that ultimately lead to oligosaccharide sequences with specific ligand binding properties. These reactions are catalyzed by GlcNAc N-deacetylase/N-sulfotransferase (NDST), a monomeric enzyme with two catalytic activities. Two genes encoding NDST isozymes have been described, one from rat liver (NDST1) and another from murine mastocytoma (NDST2). Both isozymes are expressed in tissues in varying amounts, but their relative contribution to heparan sulfate formation in any one tissue is unknown. We now report the identification of a third member of the NDST family, designated NDST3. A full-length cDNA clone (3.2 kilobase pairs) encoding a 873-amino acid protein was obtained from a human fetal/infant brain cDNA library. Human NDST3 (hNDST3) has a nucleotide sequence homologous but not identical to hNDST1 and NDST2. The deduced amino acid sequence shows 70% and 65% amino acid identity to that of hNDST1 and NDST2, respectively. A soluble chimera of hNDST3 and protein A exhibited both N-deacetylase and N-sulfotransferase activity, confirming its enzymatic identity. Northern blot analysis of human fetal brain poly(A) ؉ RNA showed a single transcript of 6.4 kilobase pairs. Reverse transcription polymerase chain reaction analysis revealed more restricted tissue expression of hNDST3 than hNDST1 and NDST2, and high levels in brain, liver, and kidney. Analysis of Chinese hamster ovary cells revealed expression of NDST1 and NDST2, but not NDST3. In a Chinese hamster ovary cell mutant exhibiting reduced N-sulfotransferase activity and reduced sulfation of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059 -8065), expression of NDST1 was greatly reduced, but NDST2 was expressed normally, suggesting that both enzymes are involved in heparan sulfate assembly. The discovery of multiple NDST isozymes suggests that the assembly of heparan sulfate is much complicated than previously appreciated.Heparan sulfate and heparin are large complex carbohydrate chains that interact with various proteins (e.g. basic fibroblast growth factor, hepatocyte growth factor, antithrombin, and lipoprotein lipase) through unique oligosaccharide sequences. These sequences consist of N-acetylated and N-sulfated glucosamine residues, containing O-sulfate groups and GlcA 1 and IdceA in various arrangements (1). The assembly of these sequences occurs by the concerted action of various enzymes that act in three stages: 1) chain initiation and assembly of the linkage tetrasaccharide GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤1-on serine residues of core protein, 2) chain elongation in which the disaccharide repeat unit of GlcA␤1,4GlcNAc␣1,4-are assembled, and 3) chain modification in which N-deacetylation/ N-sulfation of subsets of GlcNAc residues occurs, followed by C5-epimerization of GlcA to L-IdoA, 2-O-sulfation of the uronic acid residues, and 6-O-and 3-O-sulfation of glucosamine residues. GlcNAc N-deacetylat...

Endoplasmic reticulum α-1,2 mannosidase I (ERManI) is an enzyme, which removes α(1-2) linked mannoses from asparagine-linked oligosaccharides on glycoproteins in the endoplasmic reticulum (ER). ERManI preferentially removes one α(1-2) linked mannose from B-chain of Man(9)GlcNAc(2). When glycoproteins fail to achieve properly folding, increased removal of α(1-2) linked mannoses on their oligosaccharides is induced and leads them to be disposed and degraded by ER-associated degradation pathway. However, it is still inconclusive whether accelerated removal of α(1-2) linked mannoses on those glycoproteins is catalyzed by the α-1,2 mannosidase I, proteins similar to mannosidase I [e.g. ER degradation-enhancing α-1,2 mannosidase-like protein (EDEM)], or both of them. Therefore, to approach this issue, we have investigated its in vitro activities using various oligosaccharides and glycoproteins as substrates. A recombinant form of human ERManI (hERManI) was prepared by using Escherichia coli. First, the enzyme generated Man(6)GlcNAc(2)-PA and Man(5)GlcNAc(2)-PA from 100 μM Man(9)GlcNAc(2)-PA after a one-hour reaction. Second, we have exposed bovine thyroglobulin and soybean agglutinin to denaturing conditions, e.g. 8 M urea, and used those glycoproteins as substrates. Sugar moieties were released from the reactant by PNGase F and their structures and amounts were elucidated by HPLC analysis. Intriguingly, the enzyme was shown to remove mannoses from bovine thyroglobulin and soybean agglutinin to larger extents when they were exposed to a denaturant. Therefore, our results suggested that hERManI could recognize tertiary and/or quaternary structures of glycoproteins and remove more α-1,2 linked mannoses from misfolded glycoproteins in living cells.

Residue 75 on the flap, a beta hairpin loop that partially covers the active site cleft, is tyrosine in most members of the aspartic proteinase family. Site-directed mutagenesis was carried out to investigate the functional role of this residue in Rhizomucor pusillus pepsin, an aspartic proteinase with high milk-clotting activity produced by the fungus Rhizomucor pusillus. A set of mutated enzymes with replacement of the amino acid at position 75 by 17 other amino acid residues except for His and Gly was constructed and their enzymatic properties were examined. Strong activity, higher than that of the wild-type enzyme, was found in the mutant with asparagine (Tyr75Asn), while weak but distinct activity was observed in Tyr75Phe. All the other mutants showed markedly decreased or negligible activity, less than 1/1000 of that of the wild-type enzyme. Kinetic analysis of Tyr75Asn using a chromogenic synthetic oligopeptide as a substrate revealed a marked increase in kcat with slight change in K(m), resulting in a 5.6-fold increase in kcat/K(m). When differential absorption spectra upon addition of pepstatin, a specific inhibitor for aspartic proteinase, were compared between the wild-type and mutant enzymes, the wild-type enzyme and Tyr75Asn, showing strong activity, had spectra with absorption maxima at 280, 287 and 293 nm, whereas the others, showing decreased or negligible activity, had spectra with only two maxima at 282 and 288 nm. This suggests a different mode of the inhibitor binding in the latter mutants. These observations suggest a crucial role of the residue at position 75 in enhancing the catalytic efficiency through affecting the mode of substrate-binding in the aspartic proteinases.