Jean-Pierre Sine

Digital screening methodology for the directed evolution of transglycosidases

Engineering of glycosidases with efficient transglycosidases activity is an alternative to glycosyltransferases or glycosynthases for the synthesis of oligosaccharides and glycoconjugates. However, the engineering of transglycosidases by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of digital imaging-based high-throughput screening methodology for the directed evolution of glycosidases into transgalactosidases. Using this methodology, we detected transglycosidase mutants in intact Escherichia coli cells by digital imaging monitoring of the activation of non- or low-hydrolytic mutants by an acceptor substrate. We screened several libraries of mutants of beta-glycosidase from Thermus thermophilus using this methodology and found variants with up to a 70-fold overall increase in the transglycosidase/hydrolysis activity ratio. Using natural disaccharide acceptors, these transglycosidase mutants were able to synthesise trisaccharides, as a mixture of two regioisomers, with up to 76% yield.

Human intestine epithelial cell acetyl- and butyrylcholinesterase

The epithelial cells of the human intestine exhibit a cholinesterase activity which is restricted to the apex of the villi. This activity displays a maximum in the colon and a minimum in the jejunum. Contrary to most of the studied vertebrates, the human cells present both acetylcholinesterase and butyrylcholinesterase activities, acetylcholinesterase being predominant in all the intestinal segments: duodenum, jejunum, ileum and colon. Like in the other vertebrates, only globular forms are identified by sucrose gradient centrifugation. However, the simultaneous presence, on the one hand of three globular forms (G1, G2 and G4) and, on the other hand of soluble as well as detergent-soluble molecular species seems to be a particular feature of the human cells.

Acetylcholinesterase and butyrylcholinesterase in the gut mucosal cells of various mammal species: Distribution along the intestine and molecular forms

1. A cholinesterase activity was shown to be present in the homogenates of the gut mucosal cells from seven mammal species examined. 2. The distribution of the cholinesterase activity in the mucosal cells along the intestine differs from one species to another. This distribution is not correlated with that of the aminopeptidase which is a specific marker of the enterocyte plasma membranes. 3. Except rabbit, all the other species contain a (G4) globular tetrameric form and either a (G1) monomeric form (pig, ox) or a (G2) dimeric form (mouse, rat, sheep). Both (G1) and (G2) forms are found with the (G4) form in the mucosal cells of kitten and cat. The solubility characteristics of these various forms were studied by sucrose gradient centrifugations in the presence and the absence of 1% Triton X-100. 4. The mucosal cells from the studied species essentially possess either acetylcholinesterase (rabbit, kitten, cat) or butyrylcholinesterase (ox, pig, sheep, rat, mouse). These findings indicate that both enzymes probably present identical physiological functions in this cell type.

Soluble form of acetylcholinesterase from rabbit enterocytes: comparison of its molecular properties with those of the plasma membrane species

A soluble form of acetylcholinesterase was shown to be present in rabbit enterocytes. The enzyme was obtained from a high-speed supernatant (105,000 X g centrifugation) after homogenization of intestinal mucosa without detergent. It was shown to possess no obvious hydrophobic character and could be classified as a low-salt-soluble (LSS) acetylcholinesterase. Sucrose gradient centrifugation revealed a single enzyme species with a sedimentation coefficient of 3.9 +/- 0.2S. By gel filtration performed in HPLC the enzyme was eluted as a protein corresponding to an Mr of 72,000 +/- 3,000. It could be precipitated with concanavalin A by affinoelectrophoresis, but the catalytic activity was not affected by the lectin. Our results are consistent with a G1 globular form for this soluble acetylcholinesterase which differs very clearly from detergent-soluble forms also found recently in the plasma membranes of rabbit enterocytes.

Presence and characterization of acetylcholinesterase in brush-border and basolateral membranes of rabbit enterocytes

Acetylcholinesterase is found in the brush-border and basolateral membranes purified from rabbit enterocytes. The sedimentation coefficients of the enzymes solubilized from two types of membrane are identical (5.5 +/- 0.2 S) and the apparent molecular weights are not significantly different (154 000 +/- 8000 for the brush-border and 145 000 +/- 8000 for the basolateral membrane enzyme). These results suggest a unique G2 molecular form for acetylcholinesterase from brush-border as well as from basolateral membranes.

Characterization of cholinesterase molecular forms in the mucosal cells along the intestine of the chicken

The presence of a butyrylcholinesterase (BuChE, EC 3.1.1.8) in the musocal cells of the chicken intestine was demonstrated by histochemical and biochemical methods. The study of its distribution, along the intestine from duodenum to rectum, showed that the jejuno-ileum possesses the highest activity. Sucrose gradient centrifugation revealed, in all intestinal areas, two globular forms with sedimentation coefficients of 4.3 S (G1 form) and 10.8 S (G4 form). The presence of Triton X-100 in the preparations did not modify the sedimentation profiles of these two forms which can be considered as soluble BuChE. The ratio of G1/G4-forms progressively decreases along the intestine from duodenum to rectum indicating a predominance of the G4 form in the areas where the activity is low. Our results are discussed in relation to other studies of globular forms of chicken BuChE.

Electrostatic interactions of the butyrylcholinesterase dimer of mucosal cells of rat intestine with glycosaminoglycans

The G2 form of butyrylcholinesterase (BChE) of mucosal cells of rat intestine is a rare amphiphilic species, which is related to class II of acetylcholinesterase. Preliminary work indicated that the enzyme can bind heparin and suggested particular properties as compared to other BChEs. Ionic properties of the G2 form BChE were studied with different ionic exchangers. Heparin-Sepharose chromatography, nondenaturing electrophoresis and sucrose gradient centrifugation were used to study heparin interaction with the G2 form BChE. The enzyme structure was modified with reagents that react specifically with amino groups (p-hydroxyphenylglyoxal and 2,4,6-trinitrobenzene sulfonic acid). The G2 form was not retained by DEAE-cellulose which was generally used to isolate BChE from human serum, but was completely bound by strong cation exchanger (Dowex 50). Heparin-Sepharose quantitatively retained the enzyme which was partially eluted only by charged compounds. Nondenaturing gel electrophoresis showed a reduction in enzyme migration with increasing concentrations of heparin and chondroitin sulfate, but not with heparan sulfate. Triton X-100 dissociated the G2 form into monomers but failed to reverse the association between the enzyme and heparin. Reagents specific to amino groups indicated that arginine and lysine residues were involved in this association. In summary, these studies demonstrate that the ionic properties of the G2 form BChE are involved in the binding with heparin. Our results rule out the possibility of amphiphilic interactions in the formation of heparin-enzyme complex and indicate that amino groups are predominately involved in this association.

Sex-related differences in the expression of chick enterocyte butyrylcholinesterase during embryonic and post-hatching development

SummaryThe butyrylcholinesterase activity of chick enterocytes was studied from day 15 in ovo up to day 90 after hatching. The activities detected in both sexes at the level of jejuno-ileum change in a parallel manner, but the activity is always higher in the female than in the male during embryonic development. After hatching, the differences are less apparent although the study of the enzyme distribution along the intestine showed sex-related variations, mainly at the level of the anterior and middle parts of jejuno-ileum in the young adult. Analysis of butyrylcholinesterase by sucrose gradient centrifugation allowed to identify two globular soluble species (G1 and G4 forms). The G4/(G1 + G4) ratio decreases during the development but this variation in the female does not parallel that observed in the male. Besides, the molecular form distribution along the intestine, studied after hatching, differs according to the sex. Taken together our results lead to hypothesize that the ontogeny and the regulation of the chick enterocyte butyrylcholinesterase depend on hormones.

Embryonic and post-natal changes in activity and molecular forms of mucosal cell butyrylcholinesterase in chicken intestine

SummaryThe mucosal cells of the chicken intestine contain a cholinesterase activity essentially due to butyrylcholinesterase. The enzyme is present during embryonic and post-hatching development. The activity reaches a maximum value at day 19 in ovo and decreases prior to and after hatching up to day 4 ex ovo. Then the activity again rises reaching a second maximum at 2–3 weeks. Beyond this stage, the activity slowly decreases leveling off to the value determined in adult chicken. The enzyme exists as two globular forms (G1 and G4) soluble at low-ionic strengths. The G4 form is predominant in ovo up to day 19. From this stage and after hatching the G1 form is the main one. This change in the form proportion differentiates the mucosal cell butyrylcholinesterase from butyrylcholinesterase of other origins such as the chicken plasma enzyme which always shows a predominant G4 form.

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

Abstract: The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast‐twitch posterior latissimus dorsi (PLD) and in slow‐tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5‐Hz frequency (slow rhythm) or 40‐Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.