Kiichiro Totani

Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin

Lectins belonging to the jacalin‐related lectin family are distributed widely in the plant kingdom. Recently, two mannose‐specific lectins having tandem repeat‐type structures were discovered in Castanea crenata (angiosperm) and Cycas revoluta (gymnosperm). The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body. To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar‐binding specificity. For this purpose, we compared the dissociation constants (Kd) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p‐nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography. As a result, we found that the basic carbohydrate‐binding properties of CCA and CRLL were similar, but differed in their preference for larger N‐linked glycans (e.g. Man7–9 glycans). While the affinity of CCA decreased with an increase in the number of extended α1–2 mannose residues, CRLL could recognize these Man7–9 glycans with much enhanced affinity. Notably, both lectins also preserved considerable affinity for mono‐antennary, complex type N‐linked glycans, though the specificity was much broader for CCA. The information obtained here should be helpful for understanding their functions in vivo as well as for development of useful probes for animal cells. This is the first systematic approach to elucidate the fine specificities of plant lectins by means of high‐throughput, automated frontal affinity chromatography.

Effects of macromolecular crowding on glycoprotein processing enzymes.

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.

Sugar-binding activity of the MRH domain in the ER α-glucosidase II β subunit is important for efficient glucose trimming

Glucosidase II (GII) is a glycan-processing enzyme that trims two alpha1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first alpha1,3-linked glucose from Glc(2)Man(9)GlcNAc(2) (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc(1)Man(9)GlcNAc(2) (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic alpha subunit (GIIalpha) and a tightly associated beta subunit (GIIbeta) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIbeta (GIIbeta-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIbeta-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIbeta-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIbeta-MRH binds most strongly to the glycans with the alpha1,2-linked mannobiose structure. GII with the mutant GIIbeta that lost the sugar-binding activity of GIIbeta-MRH hydrolyzes p-nitrophenyl-alpha-glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIbeta-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.

Chemical approaches toward understanding glycan-mediated protein quality control.

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides during N-glycosylation, play critical roles in protein quality control. Involved in this process are a number of intracellular carbohydrate-recognizing proteins or carbohydrate-processing enzymes, including calnexin/calreticulin, malectin, glucosidase I (G-I) and II (G-II), UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (VIP36, ERGL, and ERGIC-53), ER 1,2-mannosidase I, ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) and ubiquitin ligase. Although all these proteins seem to recognize high-mannose glycans, their precise specificities are yet to be clarified. In order to conduct quantitative evaluation of the activity and specificity of these proteins, a comprehensive set of high-mannose-type glycans and their variously functionalized derivatives were synthesized and used to analyze enzymes involved in glycoprotein quality control system.

Analysis of the sugar-binding specificity of mannose-binding-type Jacalin-related lectins by frontal affinity chromatography – an approach to functional classification

The Jacalin‐related lectin (JRL) family comprises galactose‐binding‐type (gJRLs) and mannose‐binding‐type (mJRLs) lectins. Although the documented occurrence of gJRLs is confined to the family Moraceae, mJRLs are widespread in the plant kingdom. A detailed comparison of sugar‐binding specificity was made by frontal affinity chromatography to corroborate the structure–function relationships of the extended mJRL subfamily. Eight mJRLs covering a broad taxonomic range were used: Artocarpin from Artocarpus integrifolia (jackfruit, Moraceae), BanLec from Musa acuminata (banana, Musaceae), Calsepa from Calystegia sepium (hedge bindweed, Convolvulaceae), CCA from Castanea crenata (Japanese chestnut, Fagaceae), Conarva from Convolvulus arvensis (bindweed, Convolvulaceae), CRLL from Cycas revoluta (King Sago palm tree, Cycadaceae), Heltuba from Helianthus tuberosus (Jerusalem artichoke, Asteraceae) and MornigaM from Morus nigra (black mulberry, Moraceae). The result using 103 pyridylaminated glycans clearly divided the mJRLs into two major groups, each of which was further divided into two subgroups based on the preference for high‐mannose‐type N‐glycans. This criterion also applied to the binding preference for complex‐type N‐glycans. Notably, the result of cluster analysis of the amino acid sequences clearly corresponded to the above specificity classification. Thus, marked correlation between the sugar‐binding specificity of mJRLs and their phylogeny should shed light on the functional significance of JRLs.

Genetic analysis of glucosidase II β-subunit in trimming of high-mannose-type glycans

Glucosidase II (G-II) is a glycoprotein-processing enzyme that successively cleaves two alpha1,3-linked glucose residues from N-linked oligosaccharides in the endoplasmic reticulum. G-II is a heterodimer whose alpha-subunit contains a glycosidase active site, but the function(s) of the beta-subunit remain poorly defined. We report here an in vivo enzymatic analysis using gene disruptants lacking either the G-II alpha- or beta-subunit in the filamentous fungus Aspergillus oryzae. Using synthetic oligosaccharides as probes, G-II activity of the membranous fraction of the gene disruptants was investigated. The fraction lacking the beta-subunit retained hydrolytic activity toward p-nitrophenyl alpha-D-glucopyranoside but was inactive toward both Glc(2)Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2). When the fraction containing the beta-subunit was added to the one including the alpha-subunit, the glucosidase activity was restored. These results suggested that the beta-subunit confers the substrate specificity toward di- and monoglucosylated glycans on the glucose-trimming activity of the alpha-subunit.

Design and Synthesis of Oligosaccharides that Interfere with Glycoprotein Quality‐control systems

Calnexin (CNX) and its soluble homologue calreticulin (CRT) are lectin‐like molecular chaperones that help newly synthesized glycoproteins to fold correctly in the rough endoplasmic reticulum (ER). To investigate the mechanism of glycoprotein‐quality control, we have synthesized structurally defined high‐mannose‐type oligosaccharides related to this system. This paper describes the synthesis of the non‐natural undecasaccharide 2 and heptasaccharide 16, designed as potential inhibitors of the ER quality‐control system. Each possesses the key tetrasaccharide element (Glc1Man3) critical for the CNX/CRT binding, while lacking the pentamannosyl branch required for glucosidase II recognition. These oligosaccharides were evaluated for their ability to bind CRT by isothermal titration calorimetry (ITC). As expected, each of them had a significant affinity towards CRT. In addition, these compounds were shown to be resistant to glucosidase II digestion. Their activities in blocking the chaperone function of CRT were next measured by using malate dehydrogenase (MDH) as a substrate. Their inhibitory effects were shown to correlate well with their CRT‐binding affinities, both being critically dependent upon the presence of the terminal glucose (Glc) residue.

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds.

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by “free” N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N′-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Establishment of a real-time analytical method for free oligosaccharide transport from the ER to the cytosol

During N-glycosylation of proteins, significant amounts of free unconjugated glycans are also generated in the lumen of the endoplasmic reticulum (ER). These ER-derived free glycans are translocated into the cytosol by a putative transporter on the ER membrane for further processing. However, the molecular nature of the transporter remains to be determined. Here, we report the establishment of a novel assay method for free oligosaccharide transport from the ER lumen using chemically synthesized fluorescence-labeled N-glycan derivatives. In this method, fluorescence-labeled glycan substrates were encapsulated inside mouse liver microsomes, followed by incubation with the cytosol and a fluorescence-quenching agent (anti-fluorophore antibody). The rate of substrate efflux was then monitored in real time by the decrease in the fluorescence intensity. The present data clearly demonstrated that the oligosaccharide transport activity under the current assay conditions was both ATP and cytosol dependent. The transporter activity was also found to be glycan structure specific because free glucosylated glycans were unable to be transported out of the microsomes. This new assay method will be a useful tool for identifying the transporter protein on the ER membrane.