Proteolytic Cleavage of Ostrich and Turkey Pancreatic Lipases

Laboratoire de Biochimie et de Génie Enzymatique des Lipases, Ecole Nationale d'Ingénieurs de Sfax, Tunisia.
Pancreas (Impact Factor: 2.96). 11/2007; 35(3):e55-61. DOI: 10.1097/mpa.0b013e31811f450f
Source: PubMed


The aim of this study was to check some biochemical and structural properties of ostrich and turkey pancreatic lipases (OPL and TPL, respectively).
Limited proteolysis of OPL and TPL was performed in conditions similar to those reported for porcine pancreatic lipase.
In the absence of bile salts and colipase, OPL failed to catalyze the hydrolysis of pure tributyrin or efficiently hydrolyze olive oil emulsion. When bile salts and colipase were preincubated with the substrate, the OPL kinetic behavior remained linear for more than 30 minutes. The enzyme presented a penetration power value into an egg phosphatidylcholine monomolecular film that was comparable to that of HPL and lower than that of TPL. Chymotrypsin, trypsin, and thermolysin were able to hydrolyze OPL and TPL in different ways. In both cases, only N-terminal fragments accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic cleavage of OPL and TPL completely degraded the enzymes. Nevertheless, chymotryptic attack generated 35-kd and 43-kd forms for TPL and OPL, respectively. Interestingly, the OPL 43-kd form was inactive, whereas the TPL 35-kd protein conserved its lipolytic activity.
OPL, TPL, and mammal pancreatic lipases share a high amino acid sequence homology. Further investigations are, however, needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of lipases.

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Available from: Fendri Ahmed, Feb 06, 2015
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    • "The N-terminal sequence of the 35 kDa fragment was the same as the native TPL. Based on its molecular mass (35 kDa), the C-terminal truncated TPL form would correspond to the N-terminal domain which was in favour of the degradation of the C-terminal domain upon chymotryptic cleavage [9]. "
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    ABSTRACT: The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin-water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.
    PLoS ONE 08/2013; 8(8):e71605. DOI:10.1371/journal.pone.0071605 · 3.23 Impact Factor
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    • "The cleavage of TPL by chymotrypsin has generated three major fragments of about 35, 14 and 10 kDa. The N-terminal of the 35 kDa fragment was the same as the native TPL [12]. So this truncated TPL form would correspond to the TPL N-terminal domain which was active on tributyrin emulsion in the absence of colipase and in the presence of a low concentration of bile salts. "
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    ABSTRACT: Lipases share an overall alpha/beta hydrolase fold structure characteristic of serine hydrolases. Nevertheless, each lipases group possesses its characteristic 3-D structure and catalytic properties. The purified N-terminal truncated forms of a pancreatic (from ostrich) and a fungal (from Rhizopus oryzae, ROL32) (sayari et al., 2005) lipases displayed much lower activities as compared to the native proteins. The aim of this study is to explain this common functional feature on a structural basis. The molecular modelling showed that the N-terminal peptide of the fungal lipase displays an extended "V" shaped structure motif (sayari et al., 2005). We observed that the N-terminal peptide of a pancreatic lipase shares the same extended structure with that of the ROL32, despite the low sequence homology between the two peptides. Upon superimposition of the 3-D structure of the N-terminal catalytic domain of the pancreatic lipase with the model of the ROL32, we have shown that the N-terminal peptide and the open lid domain, of each lipase, are located distally within the putative interfacial binding surface. In particular, two hydrophobic residues, Leu and Ile belonging to the N-terminal peptide of each lipase are well placed to interact with the lipidic substrate. Furthermore, the N-terminal peptide of each lipase seems to be well placed to interact with the loop bearing the catalytic aspartic acid. All these observations might explain the fact that the loss of the N-terminal peptide affects the lipase activity. This work shows that the two lipases share striking structural and functional features with respect to their N-terminal peptide despite the fact that they belong to very distant kingdoms such as fungal and higher animals' ones.
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