Glycan specificity of a testis-specific lectin chaperone calmegin and effects of hydrophobic interactions.

BACKGROUND: Testis-specific chaperone calmegin is required for the generation of normal spermatozoa. Calmegin is known to be a homologue of endoplasmic reticulum (ER) residing lectin chaperone calnexin. Although functional similarity between calnexin and calmegin has been predicted, detailed information concerned with substrate recognition by calmegin, such as glycan specificity, chaperone function and binding affinity, are obscure. METHODS: In this study, biochemical properties of calmegin and calnexin were compared using synthetic glycans and glycosylated or non-glycosylated proteins as substrates. RESULTS: Whereas their amino acid sequences are quite similar to each other, a certain difference in secondary structures was indicated by circular dichroism (CD) spectrum. While both of them inhibited protein heat-aggregation to a similar extent, calnexin exhibited a higher ability to facilitate protein folding. Similarly to calnexin, calmegin preferentially recognizes monoglucosylated glycans such as Glc1Man9GlcNAc2 (G1M9). While the surface hydrophobicity of calmegin was higher than that of calnexin, calnexin showed stronger binding to substrate. We reasoned that lectin activity, in addition to hydrophobic interaction, contributes to this strong affinity between calnexin and substrate. CONCLUSIONS: Although their similarity in carbohydrate binding specificities is high, there seems to be some differences in the mode of substrate recognition between calmegin and calnexin. GENERAL SIGNIFICANCE: Properties of calmegin as a lectin-chaperone were revealed in comparison with calnexin.

Trimming of glucosylated N-glycans by human ER α1,2-mannosidase I.

In the endoplasmic reticulum (ER), folding of proteins modified by asparagine-linked (N-linked) glycosylation is precisely monitored by quality control machinery. Upon exit from the calnexin/calreticulin cycle, glycoproteins are digested by α-mannosidases in the ER, especially α1,2-mannosidase I (ERManI). ERManI removes the α1,2-linked mannose of the B-chain from properly folded ER glycoproteins, whereas two or more α1,2-linked mannose residues are sequentially trimmed from improperly folded glycoproteins so they are recognized by a complex that mediates ER-associated degradation (ERAD). We have shown that the efficiency of Man9GlcNAc2 de-mannosylation in model glycoproteins by recombinant human ERManI (hERManI) is dependent on folding status (Aikawa et al. (In vitro mannose trimming property of human ER α-1,2 mannosidase I. Glycoconj. J 2012;29: 35-45.)). In this study, we revealed that this enzyme also accepts N-linked sugar chains with glucose moieties as substrates with nearly identical reactivity. The ability of hERManI to remove mannose residues from GlcMan9GlcNAc2 in model glycoproteins, such as Aspergillus oryzae ß-galactosidase and chicken immunoglobulin Y (IgY), was markedly augmented when glycoproteins were denatured. The properties of hERManI enable rapid selection of ERAD substrates in the ER and may help maintain homeostasis of sugar metabolism in living organisms.

In vitro mannose trimming property of human ER α-1,2 mannosidase I.

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.

Caenorhabditis elegans proteins captured by immobilized Galß1-4Fuc disaccharide units: assignment of 3 annexins.

Galß1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan-protein interactions and corresponds to the Galß1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galß1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galß1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC-MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galß1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galß1-4Fuc column in the presence of Ca(2+). Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galß1-4Fuc, but no affinity toward Galß1-3Fuc and Galß1-4GlcNAc. We would hypothesize that the recognition of the Galß1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.

Ligand-binding activity and expression profile of annexins in Caenorhabditis elegans.

Mammalian annexins are implicated in several physiological mechanisms based on their calcium-dependent phospholipid/membrane binding and carbohydrate-binding activities. In this study, we investigated gene expression profiles of all four Caenorhabditis elegans annexins, nex-1, -2, -3 and -4, throughout the development, and compared phospholipid- and carbohydrate-binding properties of their protein products, NEX-1, -2, -3 and -4. We found that nex-1 and -3 are transcribed continuously during the developmental stages, while expression of nex-2 and -4 appeared to be temporal, peaking at the L1 stage followed by a gradual decrease toward the adult stage. NEX-1 and -3 were detected as single protein band in total worm extracts by immunoblotting, but NEX-2 was heterogenic in size. NEX-1, -2, and -3 showed the binding activities to phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, but not to phosphatidylcholine. In contrast to their uniform phospholipids-binding properties, their glycosaminoglycan-binding activities were distinctive. NEX-2 bound to heparan sulfate and chondroitin, NEX-3 bound only to heparan sulfate, and NEX-1 showed no lectin activities under tested conditions. NEX-4 had neither phospholipids- nor carbohydrate-binding properties. Differentiated expression profiles and ligand-binding properties of NEX-1, -2, -3 and -4, shown in our study, may represent distinctive roles for each C. elegans annexins.