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Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus

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Abstract

A gene encoding a putative sialidase was identified in the genome of the opportunistic fungal pathogen, Aspergillus fumigatus. Computational analysis showed that this protein has Asp box and FRIP domains, it was predicted to have an extracellular localization, and a mass of 42 kDa, all of which are characteristics of sialidases. Structural modeling predicted a canonical 6-bladed β-propeller structure with the model’s highly conserved catalytic residues aligning well with those of an experimentally determined sialidase structure. The gene encoding the putative Af sialidase was cloned and expressed in Escherichia coli. Enzymatic characterization found that the enzyme was able to cleave the synthetic sialic acid substrate, 4-methylumbelliferyl α-D-N-acetylneuraminic acid (MUN), and had a pH optimum of 3.5. Further kinetic characterization using 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside revealed that Af sialidase preferred α2-3-linked sialic acids over the α2-6 isomers. No trans-sialidase activity was detected. qPCR studies showed that exposure to MEM plus human serum induced expression. Purified Af sialidase released sialic acid from diverse substrates such as mucin, fetuin, epithelial cell glycans and colominic acid, though A. fumigatus was unable to use either sialic acid or colominic acid as a sole source of carbon. Phylogenetic analysis revealed that the fungal sialidases were more closely related to those of bacteria than to sialidases from other eukaryotes.

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Abbreviations

bodipy-Lac:

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-lactose

PCR:

polymerase chain reaction

RT:

reverse transcriptase

MEM:

minimal essential media

IPTG:

isopropyl β-D-thiogalactopyranoside

α2-3SMUG:

α2-3 isomer of 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside

α2-6SMUG:

α2-6 isomer of 4-methylumbelliferyl α-D-N-acetylneuraminylgalactopyranoside

MU:

4-methylumbelliferone

MUN:

4-methylumbelliferyl α-D-N-acetylneuraminic acid

Ni-NTA:

nickel nitrilotriacetic acid agarose

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

LB:

Luria Bertani media.

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Acknowledgements and funding

We are grateful to Dr. Steve Withers for the kind gifts of bodipy-lactoside, bodipy-lactose-sialic acid and the recombinant T. cruzi trans-sialidase. We acknowledge Dr. Jamie Rich and Ms. Yuan Yao for their expert technical assistance with the trans-sialidase assay. This work was supported by the Natural Sciences and Engineering Research Council of Canada (MM and AB).

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Authors and Affiliations

  1. Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada

    Mark L. Warwas, Juliana H. F. Yeung, Arne Ø. Mooers & Margo M. Moore

  2. Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada

    Deepani Indurugalla & Andrew J. Bennet

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  1. Mark L. Warwas
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  2. Juliana H. F. Yeung
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Correspondence to Margo M. Moore.

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Mark L. Warwas and Juliana H. F. Yeung contributed equally to this work.

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Warwas, M.L., Yeung, J.H.F., Indurugalla, D. et al. Cloning and characterization of a sialidase from the filamentous fungus, Aspergillus fumigatus . Glycoconj J 27, 533–548 (2010). https://doi.org/10.1007/s10719-010-9299-9

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