Anna Lupi

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Human prolidase and prolidase deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations

Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the prolidase deficiency (PD). Prolidase deficiency is a rare recessive disorder caused by mutations in the prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.

Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts.

Abstract. Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon 11 and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.

Human recombinant prolidase from eukaryotic and prokaryotic sources Expression, purification, characterization and long-term stability studies

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.

Therapeutic apheresis exchange in two patients with prolidase deficiency

Summary BackgroundProlidase deficiency is a rare genetic disorder for which a cure has not yet been found.

Identifying the structure of the active sites of human recombinant prolidase

In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (prolidase deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.

Characterization of a new PEPD allele causing prolidase deficiency in two unrelated patients: natural-occurrent mutations as a tool to investigate structure-function relationship

AbstractProlidase deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425-743) and PD4 (nt 661-973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the α2 domain of the “pita bread” structure proposed for E. coli and human prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.

Surfactant apoprotein A modulates interleukin-8 and monocyte chemotactic peptide-1 production

Previous studies have shown that surfactant apoprotein A (SP-A) and natural or synthetic surfactant can modulate the release of pro-inflammatory cytokines from alveolar mononuclear phagocytes. The aim of this study was to assess whether SP-A or Surfactant (Surf) from patients with pulmonary alveolar proteinosis (PAP) can affect the release of two chemokines (interleukin (IL)-8 and monocyte chemtactic peptide (MCP)-1) from human monocytes and rat lung type-II cells. In addition IL-8 and MCP-1 levels were assessed in the brochoalveolar lavage fluid (BALF) of seven patients with PAP and compared with those in a group of control subjects (n=5). SP-A, tested over a wide range of concentrations, significantly increased IL-8 and MCP-1 release from monocytes. SP-A retained its activity after collagenase digestion, but was not active after heat treatment. The release of IL-8 by monocytes was also stimulated by Surf. Finally, median BALF IL-8 and MCP-1 levels in PAP patients were significantly higher than in controls (9.50 and 9.51 pg·mL−1 in controls versus 151.95 and 563.70 pg·mL−1 in PAP, respectively) and significantly correlated with SP-A concentrations in BALF. Overall the results of this study support the view that the high content of alveolar surfactant apoprotein A may contribute to the upregulation of chemokine release in pulmonary alveolar proteinosis, thus contributing to airway inflammation.

α1-Antitrypsin in serum determined by capillary isoelectric focusing

A capillary isoelectric focusing (CIEF) method using bare fused‐silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5—5.0 range has been developed for the identification of alpha1‐antitrypsin (α1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose α1AT phenotype was previously identified and of twelve subjects whose α1AT phenotype was unknown. The results obtained allowed us to confirm or identify the α1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of α1AT variants associated with diseases of clinical relevance.

Effect of different antibacterial agents and surfactant protein-A (SP-A) on adherence of some respiratory pathogens to bronchial epithelial cells

Some antibiotics at sub-inhibitory concentrations are able to alter bacterial surface structures and modulate adhesiveness by affecting the expression of microbial adhesins. An important mechanism of pulmonary defence against pathogens is SP-A, one of the proteins of the alveolar surfactant having opsonizing activity. The aim of this study was to investigate the effect that sub-inhibitory concentrations of different antibiotics and physiological concentrations of SP-A (1 and 5 microg/ml) could exert on the adherence of respiratory pathogens to the bronchial epithelial cell line, WI26VA4. Cefdinir and clarithromycin showed high efficacy, mainly at 1/2 MIC, in reducing the adherence of Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae strains to values lower or equal to 50% of the control; sparfloxacin showed the same effect on S. aureus and S. pneumoniae but teicoplanin only on S. pneumoniae. Other similar results were observed with netilmicin on Klebsiella pneumoniae (40%) and with cefepime and ciprofloxacin on Pseudomonas aeruginosa (60%). Clarithromycin reduced the adherence of K. pneumoniae to 80% although it is not active against this strain. Adherence of the test strains was not modified by SP-A alone or in combination with any of the antibiotics used.