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

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.

Keywords: CES, carboxylesterases; Carboxylesterase; Deglycosylation; Glycosylation; Site directed mutagenesis; hCES; hCES, human carboxylesterases.

Fig. 1

Enzymatic deglycosylation of hCES2-10xHis with PNGase F. (A) Western Blot demonstrating size reduction upon denaturing and native deglycosylation of supernatants (25 µg of total protein per well). (B) Denaturing and native deglycosylation of purified hCES2-10xHis and staining with ProQ Emerald 300 (5 µg of total protein per well). Lanes: 1 – hCES2-10xHis under denaturing conditions, (PNGase F -); 2 – hCES2-10xHis under denaturing conditions (PNGase F +); 3 – hCES2-10xHis under native conditions (PNGase F -); 4 –hCES2-10xHis under native conditions deglycosylated (PNGase F +).

Fig. 2

Supernatant from cell cultures treated with tunicamycin (TM+) or DMSO (TM−) were harvested at 24 and 48 h. (A) Western Blot of the supernatants, 25 µg of total protein per well; (B) Relative activity of hCES2-10xHis in the presence or absence of tunicamycin. Results are the average of three determinations, error bars represent the standard deviation (n=3).

Fig. 3

Schematic representation of pCI-neo hCES2-10xHis mutant plasmids lacking one (p.Asn175Gln, p.Asn340Gln) or two (p.Asn175Gln/Asn340Gln) glycosylation sites. The coding sequence of each plasmid contained a signal peptide (white rectangle) and the 10xHis tag (light gray rectangle). Glycosylation sites are represented by the black rectangles.

Fig. 4

Glycosylation site mutants and positive control (fully glycosylated hCES2-10xHis) present in cell culture supernatants were analyzed. (A) Western blot analysis, 25 µg of total protein per well. (B) Relative activity of the mutants comparing to the positive control. Results are the average of three determinations; error bars represent the standard deviation of the triplicates. PC – positive control, hCES2-10xHis; Glyco 1 – hCES2-10xHis lacking the first glycosylation site; Glyco 2 – hCES2-10xHis lacking the second glycosylation site; Glyco 1+2 – hCES2-10xHis lacking both glycosylation sites; NC – negative control, supernatant of non transfected cells.

Fig. 5

Relative activity of glycosylation site mutants and of fully glycosylated hCES2-10xHis (PC) after being subjected to a temperature gradient (normalized to the activity observed at 37 °C in each case). Results are average of three determinations; error bars represent the standard deviation of the triplicates. PC – positive control, hCES2-10xHis; Glyco 1 – hCES2-10xHis lacking the first glycosylation site; Glyco 2 – hCES2-10xHis lacking the second glycosylation site; Glyco 1+2 – hCES2-10xHis lacking both glycosylation sites. * Indicates a statistically significant difference (P<0.05) in activity observed in the mutants in relation to the activity of the PC at the same temperature.