Protein and Peptide Letters - Volume 9, Issue 1, 2002

Growth and spread of tumors requires a variety of membrane and extracellular proteases to modify membrane integrins, dissolve the surrounding matrix and release critical growth factors from both the tumor cell surface and surrounding structures. The two major protease systems involved in this process are the matrix metalloproteases and the serine proteases. Genes and gene products for both protease systems are overexpressed in a variety of neoplasms. Thus, these enzymes serve as excellent targets for the delivery of potent cytotoxic molecules to tumors. A number of peptide toxins have been engineered to bind to tumor cells with high levels of surface proteases and their receptors including anthrax toxins, Pseudomonas exotoxin, saporin and diphtheria toxin. These recombinant fusion proteins provide a novel class of anti-cancer agents that will enter clinical trials in the next several years.

In order to clarify the potential role of cathepsin E at neutral pH, the cleavage specificity of human cathepsin E was examined at pH 7.4 toward reduced-carboxymethylated(RCm-)ribonuclease A and various bioactive and related peptides. The specificity of the enzyme at pH 7.4 was found to be considerably different from that at acidic pH preferential cleavages were observed with Arg-X and Glu-X bonds, which are not the major cleavage sites at acidic pH. Moreover, the Arg-Arg bond was found to be the most preferential site of cleavage. This unique specificity observed at pH 7.4 suggests the possibility that cathepsin E might be involved in processing and/or degradation of certain proteins and / or peptides at or near neutral pH in vivo.

α-Gliadins isolated by carboxymethylcellulose chromatography contain noncovalently bound glucose probably due to contaminating proteoglycans and to material shed from the column. Traces of carbohydrate remain strongly bound to α-gliadins even after harsh denaturation, but our results indicate α-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans.

Human beta-defensin-2 (hBD-2), a small cationic peptide, exhibits a broad range of antimicrobial activity. It has been found to play important roles in innate and adaptive immune responses against microbial invasion. For the purposes of this study, hBD-2 gene was cloned from the lesions of human condyloma acuminatum. An expression vector was constructed and transformed into E. coli. hBD-2 was expressed as a fusion protein in both the soluble and insoluble forms, which was further confirmed by western blotting analysis.

A practical and low cost system for isoelectric protein focusing (IEF) was developed. The system uses a multi-cell glass plate compatible with a common vertical one-dimensional electrophoresis chamber, dispensing specific IEF apparatus. After focusing, the IEF gels are easily recovered. The resulting two-dimensional (2-D) gels have provided efficient protein separation for different concentrations and extracts.

In this work we investigated the formation of a complex between γ-thionin SIα1 and Ca2+ using differential pulse voltammetry and MALDI-TOF / MS. A stoichiometry rate of one calcium ion per one SIα1 molecule was obtained. Kd values of 1.31 x 10-9 mol L-1, 2.63 x 10-8 mol L-1 and 2.71 x 10 -8 mol L-1 were determined for the Ca2+-SIα1 complex by three different methodologies. These findings contribute to a better understanding of the SIα1 inhibition mechanism towards insect and mammalian α-amylases.

A computerized database for synthetic antibiotic peptides, the SAPD, has been created and is available on the Internet at web address, http://oma.terkko.helsinki.fi:8080 / ∼SAPD. The SAPD is modelled on two pre-existing computer databases for naturally occurring peptide antibiotics, the Antimicrobial Sequences Database and the Peptaibol Database, and it will contain both chemical and biological information on all published synthetic antibiotic peptides.

ConBr, a D-glucose / D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a cDNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.

A D-glucose / D-mannose specific lectin from seeds of Canavalia grandiflora (ConGF) was purified by affinity chromatography on Sephadex G-50. By SDS-PAGE ConGF yielded three protein bands with apparent molecular masses of 29-30 kDa (α chain), 16-18 kDa (β fragment) and 12-13 kDa (γ fragment), like other related lectins from the genus Canavalia (Leguminosae). ConGF strongly agglutinates rabbit erythrocytes, has a high content of ASP and SER, and its N-terminal sequence (30 residues) is highly similar to the sequences of other related lectins from subtribe Diocleinae.

Earthworm fibrinolytic enzyme III-1 (EFE-III-1) was prepared to couple with cross-linking agarose activated by 1,1'-Carbonyl-diimidazole (CDI) in this study. Although the activity of the immobilized protease decreased to approximately 64% of the native enzyme, the activity of EFE-III-1 coupled with the resin activated by CDI was higher than that activated by cyanogen bromide (CNBr). The immobilized protease was experimentally demonstrated to hydrolyze IgG, albumin and creatine kinase, besides fibrin(ogen) and plasmin(ogen), suggesting that EFE had a broad substrate specificity.

eRF1a, one of the class-I release factors from ciliate Euplotes octocarinatus, has been crystallized by the vapor-diffusion method using polyethylene glycol 4000 as the precipitant at pH 7.5. The crystal belongs to space group P2 1 and the unit-cell parameters are a=90.4, b=107.9, c=114.8Å, β=94.2°. There appear to be four eRF1a molecules in the asymmetric unit.