Hélène Boze

Proline-rich salivary proteins have extended conformations.

Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1 ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1 ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (Rg=27.9 and 41.0+/-1 A respectively) and largest distances (rmax=110 and 155+/-10 A respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b=30 A, which is larger than the usual value (18 A-20 A) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is Rc=2.7+/-0.2A. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity.

Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

The ORF encoding the Debaryomyces castellii CBS 2923 phytase was isolated. The deduced 461-amino-acid sequence corresponded to a 51.2 kDa protein and contained the consensus motif (RHGXRXP) which is conserved among phytases. No signal sequence cleavage site was detected. Nine potential N-glycosylation sites have been predicted. The protein shared 21-69% sequence identities with various phytases of yeast or fungal origin. Heterologous expression of the D. castellii CBS 2923 phytase in the methylotrophic yeast Pichia pastoris was tested under both the P. pastoris inducible alcohol oxidase (AOX1) promoter and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Maximum production levels obtained were 476 U ml(-1), with the AOX1 expression system and 16.5 U ml(-1) with the GAP one. These productions corresponded to a 320-fold and a 10-fold overexpression of the protein, respectively as compared to the homologous production. The biochemical characteristics of the recombinant phytase were identical to those of the native enzyme.

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild‐type (WT) and clusterin‐null (Clu−/−) mice indicated a protective role of clusterin over‐expression in astrocytes lasting up to 90 days post‐ischemia. However, in in vitro and in vivo models of neonatal hypoxia‐ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu−/− and WT mice subjected to oxygen‐glucose deprivation (OGD). WT mice displayed a marked up‐regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post‐OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post‐OGD resistance of Clu−/− mice. The addition of biologically active recombinant forms of human clusterin for 24 h post‐OGD led to the abolishment of the ischemic tolerance in Clu−/− slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d‐2‐amino‐5‐phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA‐mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons.

Influence of oxygen limitation on glucose metabolism in Hanseniaspora uvarum K5 grown in chemostat

SummaryGrowth and metabolite formation were studied as a function of oxygen feed rate, in glucose-limited chemostat cultures of Hanseniaspora uvarum K5 at a dilution rate of 0.26 h−1. Alcoholic fermentation occured at an oxygen feed rate of 80 mmol.l−1.h−1 . Below this value, pyruvate decarboxylase and alcohol dehydrogenase were present at high levels. In contrast, activities of oxidative metabolism enzymes, pyruvate dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase, decreased.

Cloning and overexpression of a yeast phytase gene in Pichia pastoris

on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

N-glycosylation differences between wild-type and recombinant strains affect catalytic properties of two model enzymes: β-glucosidase and phosphatase

This study compares N-glycosylation of two model enzymes, β-glucosidase and phosphatase, between wildtype yeast strains and heterologous hosts, Pichia pastoris and Schizosaccharomyces pombe. It also tries to correlate glycosylation differences to enzyme catalytic properties and to distinguish which part of glycosylation could be attributed to the nature of microorganism or to the glycoprotein itself (sequence, number of glycosylation sites...).