Intracellular delivery of liposome-encapsulated prolidase in cultured fibroblasts from prolidase-deficient patients

Perugini, Paola; Hassan, K.; Genta, Ida; Modena, Tiziana; Pavanetto, F; Cetta, Giuseppe; Zanone, Chiara; Iadarola, Paolo; Asti, Annalia; Conti, Bice

doi:10.1016/j.jconrel.2004.09.013

Cited by 28 publications

(15 citation statements)

References 22 publications

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“…As shown in Fig. 4, profiles a → d, the electropherogram obtained after 6-days incubation from cellular extracts of a PD patient, was practically identical to that of controls [27] thus confirming that exogenous Fresh blood [47] Fresh blood containing Mn activated erythrocytes [48] Fresh blood [49] Fresh blood [26] Prolidase-loaded microspheres [50,51] Prolidase-loaded liposomes [27] prolidase had been successfully transferred from liposomes to fibroblasts.…”

Section: Enzyme Replacement Therapysupporting

confidence: 57%

“…The high resolving capacity of CE allowed this to be considered as the only technique applicable for the determination of prolidase activity in complex matrices. In this context we have applied CE to detect prolidase levels in skin cultured fibroblasts from PD patients that had been incubated with prolidase-loaded liposomes [27]. The heavy interferences of liposomes with classical Chinard's assay completely prevented the use of the colorimetric procedure in medium, washings and cellular extracts.…”

Section: Determination Of Prolidase Activitymentioning

confidence: 99%

“…However, given the poor efficiency of this system for delivering prolidase into fibroblasts, further experiments have been performed to evaluate the possibility of utilizing liposomes as enzyme carriers. Thus, prolidase-loaded vescicles (prepared with a 1:1 egg l,a-phosphatidylcholine type XI-E: cholesterol with distearoylphosphatidylcholine-polyethylen glycol 2000) were prepared by means of a thin-film hydration method, that allows the preservation of enzymatic activity, and incubated with fibroblasts from PD patients [27]. When prolidase activity was assayed in the culture medium of the above fibroblasts and in washings of correspondent cell-layers, it was hardly detectable thus indicating that very low amounts, if any, of enzyme were released from liposomes in the medium.…”

Section: Enzyme Replacement Therapymentioning

confidence: 99%

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The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach

Viglio¹,

Annovazzi²,

Conti³

et al. 2006

Journal of Chromatography B

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Section: Enzyme Replacement Therapysupporting

confidence: 57%

Section: Determination Of Prolidase Activitymentioning

confidence: 99%

Section: Enzyme Replacement Therapymentioning

confidence: 99%

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The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach

Viglio¹,

Annovazzi²,

Conti³

et al. 2006

Journal of Chromatography B

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“…The same approach was used to deliver the enzyme in human cultured fibroblasts from controls and patients with PD [42]: a 49% increase in prolidase activity after 3 days of incubation was detected. Recently, porcine kidney prolidase was encapsulated in liposomes to deliver the enzyme to cultured fibroblasts: the maximum release of enzyme activity was on day 6 of incubation [43].…”

Section: Discussionmentioning

confidence: 99%

Human recombinant prolidase from eukaryotic and prokaryotic sources

Lupi¹,

Torre²,

Campari

et al. 2006

The FEBS Journal

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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.

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“…However, in humans, prolidase is involved in the final stage of the degradation of endogenous and dietary protein and is particularly critical for collagen catabolism (Endo, Hata et al 1987). Mutations in the gene coding for human prolidase can cause prolidase deficiency, an autosomal recessive disorder characterized by skin lesions, mental retardation, and recurrent infections (Scriver et al 1983;Endo et al 1989;Endo and Matsuda 1991;Forlino et al 2002;Lopes et al 2002;Perugini et al 2005). …”

Section: General Properties Of Prolidases (X-pro Dipeptidases)mentioning

confidence: 99%

Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

Du¹,

Grunden²,

Tove³

2012

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Public Reporting Burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Final Report for "The Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for Optimization of Organophosphorus Nerve Agent Detoxification"Report Title ABSTRACTThe aims of this project were to structurally study and bioengineer thermostable prolidase from Pyrococcus furiosus to enable its use for oganophosphorus nerve agent detoxification. Prolidase contains one dinuclear Co metal-center/monomer and has optimal activity at 100°C, exhibiting no activity in the absence of Co2+ or at temperatures <50°C. Requirement for metal ions is characteristic of all organophosphorus nerve agent hydrolases and results from these enzymes containing dinuclear metal-centers with one tight-binding metal atom and a second loose-binding metal atom. One primary objective of this study was to determine which of the metal sites is integral and which is labile, information that will be used to bioengineer prolidases. Another objective was to produce P. furiosus prolidase mutants that have increased catalytic activity over a lower range of temperatures using random mutation and a low-temperature selection method. Three mutant prolidases targeting metal-binding amino acids have been successfully produced and biochemical analysis has demonstrated that the Co1 metal-binding site is the high-affinity site and the Co2 site, the low-affinity site. Conditions for selection of mutant prolidases with increased activity at lower temperatures have been determined and mutant prolidases (G39E and E236V) isolated that have higher activity than wild type at 37 °C.

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Intracellular delivery of liposome-encapsulated prolidase in cultured fibroblasts from prolidase-deficient patients

Cited by 28 publications

References 22 publications

The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach

The role of emerging techniques in the investigation of prolidase deficiency: From diagnosis to the development of a possible therapeutical approach

Human recombinant prolidase from eukaryotic and prokaryotic sources

Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

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