The Structure of Bovine Lysosomal α-Mannosidase Suggests a Novel Mechanism for Low-pH Activation

Heikinheimo, Pirkko; Helland, Ronny; Leiros, Hanna‐Kirsti S.; Leiros, Ingar; Karlsen, Solveig; Evjen, Gry; Ravelli, Raimond B. G.; Schoehn, Guy; Ruigrok, Rob W.H.; Tollersrud, Ole K.; McSweeney, Seán; Hough, Edward

doi:10.1016/s0022-2836(03)00172-4

Cited by 87 publications

(126 citation statements)

References 49 publications

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“…This N-terminal ␤ region has a low degree of structural similarity to Thermoactinomyces vulgaris R-47 ␣-amylase II (Protein Data Bank code 1BVZ) (43), T. maritima maltosyltransferase (Protein Data Bank code 1GJU) (44), and bovine lysosomal ␣-mannosidase (Protein Data Bank code 1O7D) (45), but the function of the N-terminal region remains unclear.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum

Nagae

Tsuchiya

Katayama

et al. 2007

Journal of Biological Chemistry

118

101

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1,2-␣-L-Fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fuc␣1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 Å resolution. The AfcA Fuc domain is composed of four regions, an N-terminal ␤ region, a helical linker, an (␣/␣) 6 helical barrel domain, and a C-terminal ␤ region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu 566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn 423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2-fucosyllactose, and its location is nearly identical near the O1 atom of ␤-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.

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Section: Resultsmentioning

confidence: 99%

Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum

Nagae

Tsuchiya

Katayama

et al. 2007

Journal of Biological Chemistry

118

101

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“…53 The observation that the wild-type dGMII does not accommodate thio-substituted glycosides remains an important but unresolved question. Evidently, there are some chemical characteristics of the catalytic site, the thioglycosidic linkage, or both that preclude binding.…”

Section: Discussionmentioning

confidence: 99%

Probing the Substrate Specificity of Golgi α-Mannosidase II by Use of Synthetic Oligosaccharides and a Catalytic Nucleophile Mutant

et al. 2008

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Inhibition of Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an α(1,3)-or α(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the α(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the α(1,6)-linked Man of the natural substrate and the β(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the β(1,2)-linked GlcNAc. The structure is consistent with the ~80-fold preference of dGMII for the cleavage of substrates containing a nonreducing β(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

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“…Secondary structure features derived from the bovine LysMan structure (Protein Data Bank code 1O7D (56)) are also indicated below the sequence alignment demonstrating a dominant conservation in sequence within regions of secondary structure. All of the members of this novel vertebrate clade also retain the conserved catalytic acid/base (Asp ) in the active sites of both bovine LysMan (56) and Drosophila Golgi ␣-mannosidase II (57) (numbering based on the novel human ␣-mannosidase sequence, colored boxes, Fig. 2).…”

Section: Isolation Of a Human Cdna Homolog Of The Pig And Mouse 135-kdamentioning

confidence: 99%

“…Both Drosophila Golgi ␣-mannosidase II and bovine LysMan are proposed to contain an enzyme-bound Zn 2ϩ ion in their respective active sites involved in direct interactions with glycone substrate hydroxyl residues (56,57). We tested the effects of various divalent cations on both the novel human ␣-mannosidase and human LysMan following removal of dissociable cations by treatment with EDTA (Fig.…”

Section: Recombinant Protein Expression In Hek293mentioning

confidence: 99%

Characterization of a Human Core-specific Lysosomal α1,6-Mannosidase Involved in N-Glycan Catabolism

Park

Stanton

et al. 2005

Journal of Biological Chemistry

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In humans and rodents, the lysosomal catabolism of core Man 3 GlcNAc 2 N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal ␣-mannosidase (LysMan), core-specific ␣1,6-mannosidase, ␤-mannosidase, and cleavage at the reducing terminus by a di-Nacetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 ␣-mannosidase with catalytic characteristics similar to those established previously for the core-specific ␣1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, and stimulation by Co 2؉ and Zn 2؉). Substrate specificity studies comparing the novel human ␣-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the ␣1-6mannose residue from Man 3 GlcNAc but not Man 3 GlcNAc 2 or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before ␣1,6-mannosidase activity. In contrast, LysMan cleaved all of the ␣-linked mannose residues from high mannose oligosaccharides except the core ␣1-6mannose residue. ␣1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine ␣1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5-flanking sequences for the bovine ␣1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and ␣1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.

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The Structure of Bovine Lysosomal α-Mannosidase Suggests a Novel Mechanism for Low-pH Activation

Cited by 87 publications

References 49 publications

Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum

Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum

Probing the Substrate Specificity of Golgi α-Mannosidase II by Use of Synthetic Oligosaccharides and a Catalytic Nucleophile Mutant

Characterization of a Human Core-specific Lysosomal α1,6-Mannosidase Involved in N-Glycan Catabolism

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