Human recombinant prolidase from eukaryotic and prokaryotic sources

Abstract

Prolidase is a Mn2+‐dependent dipeptidase that cleaves imidodipeptides containing C‐terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N‐terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz‐Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long‐term stability study of this enzyme at 37 °C was therefore undertaken. For this analysis, an ‘on‐column’ digestion of the N‐terminal His tag by Factor Xa was performed. A positive effect of Mn2+ and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

“…All the pathological variants were expressed in E.coli (hRecProl-231delY, hRecProl-E412K and hRecProl-G448R) and purified following the protocol previously optimized for the wild type enzyme [21]. The recombinant proteins revealed a single band of 57 kDa in SDS-PAGE, indicating an overall purity >95% ( Figure 1D ), and were properly recognized by a specific antibody against human prolidase ( Figure 1D ).…”

“…Mutant human recombinant prolidase forms (hRecPro-231delY, hRecProl-E412K and hRecProl-G448R) were obtained in prokaryotic host ( E. coli ) as previously described for the wild type protein (hRecProl-WT) [21]. Several protein preparations were needed to obtain a sufficient amount of hRecProl-G448R, due to a marked precipitation trend of this mutant.…”

FE(II) Is the Native Cofactor for Escherichia coli Methionine Aminopeptidase

“…Prolidase and aminopeptidase share the same five metal ligands at the active site and share the same "pita-bread" overall structural fold with MetAP (54). E. coli prolidase was reported as a Co(II) enzyme (55), while human prolidase is believed to be a Mn(II) enzyme (56). Although E. coli aminopeptidase P (57, 58) and human cytosolic homolog (59) were characterized as Mn(II) enzymes, both human and porcine membrane-bound aminopeptidase P were described as Zn(II)-containing enzymes (60,61).…”

Structural Basis of Substrate Selectivity of E. coli Prolidase

“…Such metal variability has been observed in other pita-bread fold proteins [29] , [30] . Interestingly, the human prolidase can utilize magnesium, though to significantly lower extent than manganese – a feature not commonly seen in other prolidases [2] , [4] , [28] , [31] . Crystal structures of various prolidases, particularly those with bound substrates or inhibitors, have provided important structural insights into how these enzymes bind substrate peptides and metals, though few members of this enzyme family have been thoroughly examined biochemically.…”