Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells

Abstract

For the construction of an overexpression system of the intracellular 1,2-alpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1, 2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-alpha-d-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manalpha1-3[Manalpha1-2Manalpha1-3(Manalpha1-6) Manalpha1-6]Manbeta1- 4GlcNAcbeta1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1.cm-1. Contents of the secondary structure (alpha-helix, beta-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 degrees C by differential scanning calorimetry. The calorimetric enthalpy, DeltaHcal, of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

Use of Endoglycosidase H as a diagnostic tool for MAN1B1‐CDG patients

“…As expected, the enzymatic digestion gave one major peak corresponding to the branched Man 5 GlcNAc 1 structure ( m / z 1334) (Fig. A) . However in Man1B1‐deficient patients, two major recalcitrant peaks corresponding to the glycan structures NeuAc 1 Hex 6 HexNAc 2 ( m / z 2145) and NeuAc 1 Hex 5 HexNAc 2 ( m / z 1940) are seen besides to the observed Hex 5 HexNAc 1 structure (Fig.…”

Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii 1,2‐α‐Mannosidase Identified in the Coccidioidal T27K Vaccine by Immunoproteomic Methods

“…nidulans , P . citrinum, and Trichoderma reesei and the fungal enzyme has been expressed as a recombinant protein 15 , 21–25 . The structural and enzymatic properties of the A .…”

“…saitoi , T. reesei , and P . citrinum class I 1,2‐α‐mannosidases have also been studied extensively 18,19,24,26–28 . The filamentous fungal enzymes belong to a functionally distinct subgroup of the class I enzymes that include the mammalian Golgi 1,2‐α‐mannosidases 20 .…”

Protein Glycosylation inAspergillus fumigatusIs Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development

“…Class II α -mannosidases include the lysosomal mannosidases, Golgi mannosidase II, yeast vascular mannosidase [98, 99], and ER α -mannosidase II [122, 123]. Several Golgi α -mannosidases have been cloned and characterized from Penicillium citrinum [124, 125] , A. saitoi [126] , A. oryzae [127] , T. reesei [128], and A. nidulans [102, 129]. These enzymes are all monomeric with a molecular weight of 50–60 kDa and show the maximal activity in the semiacidic condition (pH 4–6).…”