Structural and dynamical properties of KTS-disintegrins: A comparison between Obtustatin and Lebestatin.

Obtustatin and Lebestatin are lysine-threonine-serine (KTS)-disintegrins, which are a family of low molecular weight polypeptides present in many viperidae venoms and are potent and specific inhibitors of collagen-binding integrins. The integrin binding loop, harboring the (21)KTS(23) motif, and the C-terminal tail are known to be responsible for the selective binding to the α1ß1 integrin. Despite a very high sequence homology (only two mutations are present in Lebestatin relative to Obtustatin, namely R24L and S38L), Lebestatin exhibits a higher inhibitory effect than Obtustatin on cell adhesion and cell migration to collagens I and IV. Here we show, by means of molecular dynamics simulations of the two polypeptides in aqueous solution, that Lebestatin possesses a higher flexibility of the C-terminal tail and a greater solvent accessibility of the integrin binding loop than Obtustatin. It may be hypothesized that these properties may contribute to the higher binding-affinity of Lebestatin to its biological partner.

Structural characterization of a new statherin from pig parotid granules.

This study describes the identification and structural characterization of Sus scrofa statherin. HPLC-electrospray ionization mass spectrometry analysis on pig parotid secretory granule extracts evidenced a peptide with a molecular mass value of 5381.1 +/- 0.6 Da and its truncated form, devoid of the C-terminal Ala residue, with a molecular mass value of 5310.1 +/- 0.6 Da. The complete sequence of pig statherin gene was determined by sequencing the full-length cDNA obtained by rapid amplification of cDNA ends. The gene is 549 base pairs long and contains an open reading frame of 185 nucleotides, encoding a 42-amino acid secretory polypeptide with a signal peptide of 19 residues. This sequence presents some typical features of the four statherins characterized till now, showing the highest degree of amino acid identity with bovine (57%) and human statherin (39%). Pig statherin is mono-phoshorylated on Ser-3, while primate statherins already characterized are di-phosphorylated on Ser-2 and Ser-3. This difference, probably connected to the Asp-4 --> Glu substitution, suggests the involvement of the Golgi-casein kinase, which strictly recognizes the SX(E/pS) consensus sequence.

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea.

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins.

Antifungal-protein production in maize (Zea mays) suspension cultures.

The growing emergency due to the phenomenon of drug resistance to micro-organisms has pushed forward the search for new potential drug alternatives to those already in use. Plants represent a suitable source of new antifungal molecules, as they produce a series of defensive proteins. Among them are the PRPs (pathogenesis-related proteins), shown to be effective in vitro against human pathogens. An optimized and established cell-suspension culture of maize (Zea mays) was shown to constitutively secrete in the medium a series of PRPs comprising the antifungal protein zeamatin (P33679) with a final yield of approx. 3 mg/litre. The in-vitro-produced zeamatin possessed antifungal activity towards a clinical strain of the human pathogenic yeast Candida albicans, an activity comparable with the one reported for the same protein extracted from maize seeds. Along with zeamatin, other PRPs were expressed: a 9 kDa lipid-transfer protein, a 26 kDa xylanase inhibitor and a new antifungal protein, PR-5. A fast, two-step chromatographic procedure was set up allowing the complete purification of the proteins considered, making this cell line a valuable system for the production of potential antifungal agents in a reliable and easy way.

Mass spectrometry strategies applied to the characterization of proline-rich peptides from secretory parotid granules of pig (Sus scrofa).

Basic proline-rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline-rich peptides (SP-A and SP-B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss-Prot data bank: Q95JC9-1, Q95JC9-2 and Q95JC9-3). Together the coding regions for SP-A and SP-B, which are repeated many times, account for 52-70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP-HPLC-ESI-IT-MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline-rich peptides derived from the three proproteins. Together with the coding regions for SP-A and SP-B already identified it was possible to assign 68-75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases.

Pro-oxidant activity of histatin 5 related Cu(II)-model peptide probed by mass spectrometry.

Histatin 5 is a cationic salivary peptide with strong candidacidal and bactericidal activity at physiological concentration. In this paper we demonstrate by optical spectroscopy and ESI-IT-MS experiments that a synthetic peptide related to the N-terminus of histatin 5 specifically binds copper ions in vitro and that the complex metal-peptide generates reactive oxygen species at physiological concentration of ascorbate, leading to significant auto-oxidation of the peptide within short reaction time. The oxidative activity of this peptide is associated to the presence of a specific metal binding site present at its N-terminus. The motif is constituted by the amino acid sequence NH(2)-Asp-Ser-His, representing a copper and nickel amino terminal binding site, known as "ATCUN motif". The results of the study suggest that the production of reactive oxygen species can be an intrinsic property of histatin 5 connected to its ability to bind metals.

HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivary statherin generated by transglutaminase 2.

In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (M(av)5380.0 +/- 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 +/- 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique Ione-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (> 95%), with the percentage of Gln-39 implicated in the cross-linkiing being less than 5%. The main derivative was named cyclostatherin Q37. Guineapig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 +/- 0.06 microM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".

Functional and structural characterization of the myoglobin from the polychaete Ophelia bicornis.

The myoglobin of the polychaete annelid Ophelia bicornis was isolated, purified to homogeneity and characterized. The primary structure, obtained from cDNA and protein sequencing, consists of 139 amino acid residues. The alignment with other globin sequences showed that O. bicornis myoglobin misses the pre-A helix and the first six residues of the A helix. The presence of a PheB10-GlnE7 haem distal residue pair is in agreement with the measured oxygen affinity (P50=0.85 mmHg; 1 mmHg=0.133 kPa) and the only slightly higher autoxidation rate constant (0.28 h(-1)) with respect to that of the sperm whale myoglobin mutant E7 His-->Gln (0.21 h(-1)) and to elephant myoglobin (0.1 h(-1)). Oxygen-binding co-operativity was found to be absent under all the examined experimental conditions. The resistance of O. bicornis myoglobin towards autoxidation seems to confirm the important role of part of the A helix in the stability of the globin. The higher pKa of the acid-alkaline ferric transition of O. bicornis with respect to Asian elephant myoglobin, as well as the higher absorbance ratio of its ferric form to the oxy form measured in the Soret region (gammamet/gammaoxy) with respect to that of the African elephant myoglobin, suggested a stronger interaction between the distal glutamine and the water molecule at the sixth co-ordinate position.

Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry.

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.

Polar bear hemoglobin and human Hb A0: same 2,3-diphosphoglycerate binding site but asymmetry of the binding?

Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.