Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway.

Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-mannosidases, including endoplasmic reticulum (ER) α-mannosidase I (ERManI) and Golgi α-mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme-substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.

High-throughput multimodal strong anion exchange purification and N-glycan characterization of endogenous glycoprotein expressed in glycoengineered Pichia pastoris.

The secretory pathway of the yeast Pichia pastoris has been engineered to produce complex human-type N-glycans (Choi et al., Proc Natl Acad Sci USA 100:5022-5027, 2003; Hamilton et al., Science 301:1244-1246, 2003; Hamilton et al., Science 313:1441-1443, 2006). In contrast to the heterogeneous glycans produced on the therapeutic glycoproteins expressed in mammalian cell lines, glycoengineered P. pastoris can be designed to produce a specific, preselected glycoform. In order to achieve glycan uniformity on the target protein, No Open Reading Frame (NORF) yeast cell lines are screened extensively during various stages of glycoengineering. In the absence of the target protein of interest, screening the NORF yeast cell lines for glycoform uniformity becomes a challenge. The common approach so far has been to analyze the total cell glycan pool released from glycoproteins of the NORF yeast cells to predict the N-glycan uniformity. As this does not always accurately predict the N-glycan end product, we describe in this chapter a detailed protocol for a non-affinity-based high-throughput purification of an endogenous glycoprotein. This protein of interest has been introduced during the early stages of glycoengineering process and its N-glycan profile is utilized as a tool for glycoengineering screening.

Elimination of ß-mannose glycan structures in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing ß-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the ß-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of ß-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of ß-Man-associated glycans and their related antigenicity. Taken together, the elimination of ß-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

A practical synthesis of kifunensine analogues as inhibitors of endoplasmic reticulum alpha-mannosidase I.

[reaction: see text] A practical synthesis of the potent class I alpha-mannosidase inhibitor kifunensine (1) beginning from the inexpensive and readily available starting material L-ascorbic acid (15) is described. The protected amino-alcohol ((2R,3R,4R,5R)-5-amino-2,3:4,6-diisopropylidenedioxyhexanol, 11) served as a key intermediate from which several N-1 substituted kifunensine analogues (including N-methyl, N-cyclohexyl, and N-bis(hydroxymethyl)methyl) and 2-desoxakifunensine analogues (including N-H and N-methyl) were prepared and screened for inhibition of human endoplasmic reticulum alpha-mannosidase I (ER Man I) and mouse Golgi alpha-mannosidase IA (Golgi Man IA). In addition, several pseudodisaccharide kifunensine analogues in which a mannose residue was tethered to N-1 of kifunensine via a two-, three-, or four-carbon linker and an affinity-bound kifunensine analogue were also prepared and evaluated for biological activity. While the synthesized N-1 kifunesine analogues were found to be less potent inhibitors of Class I alpha-mannosidases than kifuensine itself, the bis(hydroxymethyl)methylkifunensine analogue 6 was shown to selectively inhibit ER Man I over Golgi Man IA.

Energetics of substrate binding and catalysis by class 1 (glycosylhydrolase family 47) alpha-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.

Nascent glycoproteins are subject to quality control in the lumen of the endoplasmic reticulum (ER) where they can either be effectively folded with the aid of a collection of ER chaperones or they can be targeted for disposal in a process known as ER-associated degradation. Initiation of the ER disposal process involves selective trimming of N-glycans by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER alpha-mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca(2+) ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man(9)GlcNAc(2) substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 mannosidases involved in glycan maturation and ER-associated glycoprotein degradation.

Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.

Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel (3)S(1) sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the (1)C(4) conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the -1 subsite. A novel (3)H(4) conformation is proposed as the exploded transition state.

Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins.

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.

Structure of mouse Golgi alpha-mannosidase IA reveals the molecular basis for substrate specificity among class 1 (family 47 glycosylhydrolase) alpha1,2-mannosidases.

Three subfamilies of mammalian Class 1 processing alpha1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P2(1)2(1)2(1)), and the structure was solved to 1.5-A resolution by molecular replacement. The enzyme assumes an (alphaalpha)(7) barrel structure with a Ca(2+) ion coordinated at the base of the barrel similar to other Class 1 mannosidases. Critical residues within the barrel structure that coordinate the Ca(2+) ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man(6)GlcNAc(2) oligosaccharide attached to Asn(515) in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.

Crystallization and preliminary X-ray diffraction analysis of lectin-1 from Pseudomonas aeruginosa.

Carbohydrate recognition plays a role in the pathogenesis of Pseudomonas aeruginosa, a common cause of opportunistic infection in humans. Crystals of a carbohydrate-binding protein from P. aeruginosa, lectin PA-1, have now been obtained. The crystals belong to space group I222, with unit-cell parameters a = 40.25, b = 72.30, c = 133.82 A, and diffract to beyond 1.9 A resolution on a rotating-anode X-ray source. Details of crystal-growth conditions, diffraction data collection and processing are reported.