Use of SILAC for exploring sheddase and matrix degradation of fibroblasts in culture by the PIII SVMP atrolysin A: identification of two novel substrates with functional relevance.

Snake venom metalloproteinases (SVMPs) in Viperid venoms primarily function to give rise to local and systemic hemorrhage following snake envenomation. Years of research on these toxins, both in vitro and in vivo, indicate that they function by disrupting capillary basement membranes, stromal matrix and cell-cell and cell-matrix contacts to allow escape of capillary contents under pressure. However, most of these studies used either defined substrates in vitro or were limited by relevant antibodies for detection of sites of action in vivo. In this investigation we use stable isotope-labeled amino acids in culture (SILAC) to determine novel proteolytic activities for exogenously added atrolysin A, a hemorrhagic PIII SVMP isolated from Crotalus atrox venom. When comparing the solubilized products of SILAC-labeled cultured human fibroblasts treated with atrolysin A to that of untreated fibroblasts using LC/MS/MS, several proteins were identified as being released into the culture media specifically due to atrolysin A proteolytic activity. These included collagen VI, fibronectin, fibulin 2 and annexin V. Of particular interest was the observation of collagen VI and annexin V in that the release of these substrates could play a role in altering hemostasis and promote hemorrhage caused by the more typical actions of atrolysin A. In summary, this study demonstrates the utility of SILAC for exploring sheddase activity with cells in culture and suggests the presence of two novel substrates for SVMPs that may play a pathological role in altering host hemostasis during envenomation.

BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action.

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.

A second putative mRNA binding site on the Escherichia coli ribosome.

Translation in bacteria is initiated by a base-pairing interaction between the extreme 3'-end of the small-subunit rRNA and a purine-rich domain (Shine-Dalgarno (SD) sequence) preceding the initiation codon at the 5'-end of most bacterial mRNAs. Here, we describe the identification of a second functional and alternative site on the Escherichia coli ribosome which is capable of interacting with mRNA devoid of SD sequences and initiate the translation. This site is localized between nt 1340 and 1360 of the 16S rRNA in E. coli and is complementary to the untranslated region at the 5'-end of tobacco mosaic virus RNA (omega sequence).

Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E. coli.

The AGG and AGA are the least used arginine codons in E. coli but they are the most preferable ones in eukaryotes. The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA(AGG) (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992). The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes. The two tandems of AGG codons (Arg12 Arg13 and Arg163 Arg164) of hIF alpha 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site. The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.

Efficiency of the 5'-terminal sequence (omega) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli.

Recent studies have demonstrated that the 5' leader (omega sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes. In order to estimate the efficiency of omega to initiate translation of mRNA in Escherichia coli, in comparison to the Shine-Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E. coli expression vectors containing one constitutive promoter (P1) but different translation-initiation sites (S/D or omega delta 3 sequence, respectively). The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA. We report that substitution of omega delta 3 for S/D decreases the yield of expressed protein 4-1900-fold and the content of gene-specific mRNA is decreased by about sevenfold. However, in comparison with the S/D sequence, the level of protein expressed under the translational control of omega delta 3 is less sensitive to changes in the 5' coding region. We also report that the omega sequence contains a region of 10-12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E. coli, Eikenella corrodens and Xenopus laevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.

Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli.

It has been shown that tandems of rare arginine codons AGG have a strong inhibitory effect on translation of mRNA in E. coli [5]. This has been explained by the rate-limiting interaction of these codons with the less abundant tRNA(AGG) [6]. In this study tandemly repeated AGG triplets were introduced into the chloramphenicol acetyltransferase (CAT) gene either upstream of the initiation ATG codon or downstream of it (both in frame and out of frame) and the expression of the modified genes was investigated. We report that the addition of AGG clusters resulted in a substantial inhibitory effect on CAT gene expression independently of their localization in mRNA. This inhibitory effect is explained by a competition of the tandem AGGAGG with the natural Shine-Dalgarno (SD) sequence (consensus AAGGAGGU) for the 3'-end of the 16S small ribosomal RNA (rRNA).

Sequence analysis of putative E3 and fiber genomic regions of two strains of canine adenovirus type 1.

The nucleotide sequences of the genomes of two strains of canine adenovirus, type 1 (Glaxo and CLL) have been determined within the region spanning approximately 0.80-0.91 map unit. Homology comparisons with other adenoviral DNAs indicate that this region contains part of the hexon-associated protein (pVIII) precursor gene on the left, and a 57-kDa fiber-encoding gene on the right. In both viral strains, the sequences of these genes are identical. Accepting the generally highly conserved nature of the genetic organization of adenoviral genomes, the 1136-bp sequence between them would include the canine equivalent of the human adenoviral E3 region. In the Glaxo strain, which is nonattenuated, this sequence contains only two R-strand open reading frames capable of coding for polypeptides of 60 residues or more. One of these appears to code for a transmembrane glycoprotein of 194 amino acid residues. Neither ORF, however, shows significant homology with any other known adenoviral DNA or protein sequence. The corresponding region in the attenuated strain CLL is almost identical with that of the Glaxo, but with a 325-bp deletion resulting in a fusion of the two ORFs. It seems, therefore, that neither ORF is essential for viral replication in vitro, since the CLL strain replicates just as efficiently as the Glaxo. The suggestion is made that the attenuation of the CLL strain may be related to the nonfunctionality of its E3 gene product(s).