Human carboxylesterase 2: Studies on the role of glycosylation for enzymatic activity

Márcia Alves, Joana Lamego, Tiago Bandeiras, Rute Castro, Hélio Tomás, Ana Sofia Coroadinha, Júlia Costa, Ana Luisa Simplício

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation.Partial or non-glycosylated forms of a secreted form of hCES2 have been obtained by three approaches: (i) enzymatic deglycosylation with peptide N-glycosidase F; (ii) incubation with the inhibitor tunicamycin; ii) site directed mutagenesis of each or both N-glycosylation sites.Deglycosylated protein did not show a detectable decrease in enzyme activity. On the other hand, tunicamycin led to decreased levels of secreted hCES2 but the enzyme was still active. In agreement, mutation of each and both N-glycosylation sites led to decreased levels of secreted active hCES2. However, the thermostability of the glycosylation mutants was decreased.The results indicated that glycans are involved, to some extent in protein folding in vivo, however, removal of glycans does not abrogate the activity of secreted hCES2.

Original languageEnglish
Pages (from-to)105-110
Number of pages6
JournalBiochemistry and Biophysics Reports
Volume5
DOIs
Publication statusPublished - 1 Mar 2016

Keywords

  • Carboxylesterase
  • Deglycosylation
  • Glycosylation
  • HCES
  • Site directed mutagenesis

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