Characterization and subcellular localization of human neutral class II -mannosidase cytosolic enzymes/free oligosaccharides/glycoside hydrolase family 38/M2C1/N-glycosylation

Elina Kuokkanen, Wesley Smith, Marika Mäkinen, Heidi Tuominen, Maija Puhka, Eija Jokitalo, Sandrine Duvet, Thomas Berg, Pirkko Heikinheimo

Research output: Contribution to journalArticleScientificpeer-review

Abstract

A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral a-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe 21 than CO2+. Without activating cations the main reaction product was Man(8)GlcNAc, and Cu2+ completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.
Original languageEnglish
JournalGlycobiology
Volume17
Issue number10
Pages (from-to)1084-1093
Number of pages10
ISSN0959-6658
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

Cite this

Kuokkanen, Elina ; Smith, Wesley ; Mäkinen, Marika ; Tuominen, Heidi ; Puhka, Maija ; Jokitalo, Eija ; Duvet, Sandrine ; Berg, Thomas ; Heikinheimo, Pirkko. / Characterization and subcellular localization of human neutral class II -mannosidase cytosolic enzymes/free oligosaccharides/glycoside hydrolase family 38/M2C1/N-glycosylation. In: Glycobiology. 2007 ; Vol. 17, No. 10. pp. 1084-1093.
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abstract = "A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral a-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe 21 than CO2+. Without activating cations the main reaction product was Man(8)GlcNAc, and Cu2+ completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.",
author = "Elina Kuokkanen and Wesley Smith and Marika M{\"a}kinen and Heidi Tuominen and Maija Puhka and Eija Jokitalo and Sandrine Duvet and Thomas Berg and Pirkko Heikinheimo",
year = "2007",
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Characterization and subcellular localization of human neutral class II -mannosidase cytosolic enzymes/free oligosaccharides/glycoside hydrolase family 38/M2C1/N-glycosylation. / Kuokkanen, Elina; Smith, Wesley; Mäkinen, Marika; Tuominen, Heidi; Puhka, Maija; Jokitalo, Eija; Duvet, Sandrine; Berg, Thomas; Heikinheimo, Pirkko.

In: Glycobiology, Vol. 17, No. 10, 2007, p. 1084-1093.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Characterization and subcellular localization of human neutral class II -mannosidase cytosolic enzymes/free oligosaccharides/glycoside hydrolase family 38/M2C1/N-glycosylation

AU - Kuokkanen, Elina

AU - Smith, Wesley

AU - Mäkinen, Marika

AU - Tuominen, Heidi

AU - Puhka, Maija

AU - Jokitalo, Eija

AU - Duvet, Sandrine

AU - Berg, Thomas

AU - Heikinheimo, Pirkko

PY - 2007

Y1 - 2007

N2 - A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral a-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe 21 than CO2+. Without activating cations the main reaction product was Man(8)GlcNAc, and Cu2+ completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.

AB - A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral a-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe2+, Co2+, and Mn2+, and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe 21 than CO2+. Without activating cations the main reaction product was Man(8)GlcNAc, and Cu2+ completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.

U2 - 10.1093/glycob/cwm083

DO - 10.1093/glycob/cwm083

M3 - Article

VL - 17

SP - 1084

EP - 1093

JO - Glycobiology

JF - Glycobiology

SN - 0959-6658

IS - 10

ER -