Yukiko Kamiya

Defining the Glycan Destruction Signal for Endoplasmic Reticulum-Associated Degradation

The endoplasmic reticulum (ER) must target potentially toxic misfolded proteins for retrotranslocation and proteasomal degradation while avoiding destruction of productive folding intermediates. For luminal proteins, this discrimination typically depends not only on the folding status of a polypeptide, but also on its glycosylation state. Two putative sugar binding proteins, Htm1p and Yos9p, are required for degradation of misfolded glycoproteins, but the nature of the glycan degradation signal and how such signals are generated and decoded remains unclear. Here we characterize Yos9ps oligosaccharide-binding specificity and find that it recognizes glycans containing terminal alpha1,6-linked mannose residues. We also provide evidence in vivo that a terminal alpha1,6-linked mannose-containing oligosaccharide is required for degradation and that Htm1p acts upstream of Yos9p to mediate the generation of such sugars. This strategy of marking potential substrates by Htm1p and decoding the signal by Yos9p is well suited to provide a proofreading mechanism that enhances substrate specificity.

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.

Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca2+-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178–37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.

EDEM1 accelerates the trimming of α1,2-linked mannose on the C branch of N-glycans

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of ∼6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

The role of MRH domain-containing lectins in ERAD.

The endoplasmic reticulum (ER) quality control system ensures that newly synthesized proteins in the early secretory pathway are in the correct conformation. Polypeptides that have failed to fold into native conformers are subsequently retrotranslocated and degraded by the cytosolic ubiquitin-proteasome system, a process known as endoplasmic reticulum-associated degradation (ERAD). Most of the polypeptides that enter the ER are modified by the addition of N-linked oligosaccharides, and quality control of these glycoproteins is assisted by lectins that recognize specific sugar moieties and molecular chaperones that recognize unfolded proteins, resulting in proper protein folding and ERAD substrate selection. In Saccharomyces cerevisiae, Yos9p, a lectin that contains a mannose 6-phosphate receptor homology (MRH) domain, was identified as an important component of ERAD. Yos9p was shown to associate with the membrane-embedded ubiquitin ligase complex, Hrd1p-Hrd3p, and provide a proofreading mechanism for ERAD. Meanwhile, the function of the mammalian homologues of Yos9p, OS-9 and XTP3-B remained elusive until recently. Recent studies have determined that both OS-9 and XTP3-B are ER resident proteins that associate with the HRD1-SEL1L ubiquitin ligase complex and are important for the regulation of ERAD. Moreover, recent studies have identified the N-glycan species with which both yeast Yos9p and mammalian OS-9 associate as M7A, a Man(7)GlcNAc(2) isomer that lacks the alpha1,2-linked terminal mannose from both the B and C branches. M7A has since been demonstrated to be a degradation signal in both yeast and mammals.

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

The interaction of Ero1-α and PDI facilitates the electron transfer function of Ero1-α, activating a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases.

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

All three mammalian EDEM family members possess mannosidase activity and are necessary for glycoprotein degradation, but EDEM2 performs a unique, rate-limiting, first mannose trimming step upstream of EDEM1 and EDEM3.

Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells ☆

BACKGROUND N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway. SCOPE OF REVIEW This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins. MAJOR CONCLUSIONS Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway. GENERAL SIGNIFICANCE Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.

Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 1∶1 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.