Venómica. Mecanismos molecular y evolutivos de la diversificación estructural de la familia de las disintegrinas.
Venoms of Viperidae and Crotalidae snakes contain proteins that interfere with the coagulation cascade, the haemostatic system and tissue repair. The general aim of this thesis was elucidate the molecular details of the mechanisms leading to snake venom toxin diversification. We describe the application of the proteomic characterization of Echis ocellatus (Eo), Bitis arietans (Ba) and Sistrurus m. barbouri (Smb) venoms. Their protein composition was analyzed by RP-HPLC and SDS-PAGE of each separated protein fraction. The molecular mass and the number of cysteine residues/disulphide bonds were determined by MALDI-TOF mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion, peptide mass fingerprinting and de novo sequencing by MS/MS. Our results show that snake venoms are composed of proteins belonging to a few protein families, including enzymes (serine proteinases, Zn2+-dependent PI-PIV metalloproteases, group II phospholipase A2 isoenzymes) and proteins with no enzymatic activity as disintegrins, which are potent antagonist of integrins. To understand the genomic basis of the accelerated evolution of disintegrins we have undertaken the analysis of cDNAs encoding disintegrins from venom gland libraries of Eo and Ba and the genomic organization of disintegrin genes. The cDNA sequences coding for dimeric disintegrins lack the metalloproteinase domain and belong thus to the short-coding class. Analysis of two distinct messengers coding for the short disintegrin ocellatusin strongly argues for a common ancestry of short- and dimeric disintegrin subunits. We also report the cloning and sequence analysis of a novel and unique ECD-disintegrin-like domain from Ba. It contains the 16 cysteine residues that are conserved in all PIII disintegrin-like domains but lacks the cysteine-rich domain. On the other hand, comparison of the exon-intron organization revealed that the evolutionary pathway leading to the diversification of disintegrins also involved the miniminization of the gene organization by the successive removal of introns.
Document Full Text
Advisor:Calvete Chornet, Juan José; Sanz Sanz, Libia
School:Universitat de València
Source Type:Master's Thesis
Keywords:bioquímica i biologia molecular
Date of Publication:03/08/2007