Protein and Peptide Letters, Vol. 9, No. 1, 2002

Mini Review

Peptide Toxins Directed at the Matrix Dissolution Systems of Cancer Cells Pp. 1-14

Arthur E. Frankel,Thomas H. Bugge,Shihui Liu,Daniel A. Vallera,And Stephen H. Leppla

Regular Papers

Distinct Cleavage Specificity of Human Cathepsin E at Neutral pH with Special Preference for Arg-Arg Bonds Pp. 15-22

Senarath B. P. Athauda and Kenji Takahashi

[Abstract]

Are a-Gliadins Glycosylated? Pp. 23-29

J. B. Turner, G. V. Garner, D. B. Gordon, S. J. Brookes and C. A. Smith

[Abstract]

Cloning and Expression of Human Beta-Defensin-2 Gene in Escherichia Coli Pp. 31-37

Xiangming Fang, Li Peng, Zhinan Xu, Jinmin Wu,Peilin Cen

[Abstract]

Two-Dimensional Protein Analysis by Isoelectric Focusing in a Multi-Cell Plate System Pp.39-43

Beatriz dos Santos Ferreira, Alan Trindade Branco, Karlla Salim de Queiroz and Gonçalo Apolinario de Souza Filho

[Abstract]

Ca(II) -g-Thionin Complex: Interaction Studies by Differential Pulse Voltammetry and Maldi-Tof/Ms Pp. 45-52

Clarissa Silva Pires de Castro, Jurandir Rodrigues DeSouza and Carlos Bloch Jْnior

[Abstract]

Synthetic Antibiotic Peptides Database Pp. 53-57

David Wade and Jukka Englund

[Abstract]

Expression and Purification of the Recombinant Conbr (Canavalia Brasiliensis Lectin) Produced in Escherichia Coli Cells Pp. 59-66

Nadia A. P. Nogueira, Moema B. Grangeiro, Rodrigo M. S. da Cunha, Marcio V. Ramos, Maria A. O. , Alves, Edson H. Teixeira, Manoel Barral-Netto, Juan J. Calvete, Benildo S. Cavada and Thalles B. Grangeiro

[Abstract]

Purification and Partial Characterization of a Lectin from Canavalia Grandiflora Benth. Seeds. Pp. 67-73

V. M. Ceccatto B. S. Cavada , E. P. Nunes , N. A. P. Nogueira , M. B. Grangeiro , F. B. M. B. Moreno , E. H. Teixeira , A. H. Sampaio, M. A. O. Alves ;, M. V. Ramos , J. J. Calvete , and T. B. Grangeiro

[Abstract]

Immobilized Earthworm Fibrinolytic Enzyme Iii-1 with Carbonyldiimidazole Activated-Agarose Pp. 75-80

Xue-Qin Wu, Cen Wu and Rong-Qiao He

[Abstract]

Crystallization Report

Preliminary X-Ray Crystallographic Analysis of Ciliate Euplotes Octocarinatus Release Factor eRF1a Pp. 81-85

Zhang S., Zhou W., Ding Y., He X., Zhang H., Gao F., Heckmann K., Liang A & Rao Z.

[Abstract]

Abstracts

[Back to top] Peptide Toxins Directed at the Matrix Dissolution Systems of Cancer Cells

Arthur E. Frankel,Thomas H. Bugge,Shihui Liu,Daniel A. Vallera,And Stephen H. Leppla

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.

[Back to top] Distinct Cleavage Specificity of Human Cathepsin E at Neutral pH with Special Preference for Arg-Arg Bonds

Senarath B. P. Athauda and Kenji Takahashi

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.

[Back to top] Are a-Gliadins Glycosylated?

J. B. Turner, G. V. Garner, D. B. Gordon, S. J. Brookes and C. A. Smith

a-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 a-gliadins even after harsh denaturation, but our results indicate a-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans..

[Back to top] Cloning and Expression of Human Beta-Defensin-2 Gene in Escherichia Coli

Xiangming Fang, Li Peng, Zhinan Xu, Jinmin Wu,Peilin Cen

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.

[Back to top] Two-Dimensional Protein Analysis by Isoelectric Focusing in a Multi-Cell Plate System

Beatriz dos Santos Ferreira, Alan Trindade Branco, Karlla Salim de Queiroz and Gonçalo Apolinario de Souza Filho

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.

[Back to top] Ca(II) -g-Thionin Complex: Interaction Studies by Differential Pulse Voltammetry and Maldi-Tof/Ms

Clarissa Silva Pires de Castro, Jurandir Rodrigues DeSouza and Carlos Bloch Jْnior

In this work we investigated the formation of a complex between g -thionin SI a1 and Ca²⁺ using differential pulse voltammetry and MALDI TOF/MS. A stoichiometry rate of one calcium ion per one SI a1 molecule was obtained. K_d 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⁺ - SI a1 complex by three different methodologies. These findings contribute to a better understanding of the SI a1 inhibition mechanism towards insect and mammalian a-amylases.

[Back to top] Synthetic Antibiotic Peptides Database

David Wade and Jukka Englund

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.

[Back to top] Expression and Purification of the Recombinant Conbr (Canavalia Brasiliensis Lectin) Produced in Escherichia Coli Cells

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.

[Back to top] Purification and Partial Characterization of a Lectin from Canavalia Grandiflora Benth. Seeds.

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 (a chain), 16-18 kDa (b fragment) and 12-13 kDa (g 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.

[Back to top] Immobilized Earthworm Fibrinolytic Enzyme Iii-1 with Carbonyldiimidazole Activated-Agarose

Xue-Qin Wu, Cen Wu and Rong-qiao He

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.

[Back to top] Preliminary X-Ray Crystallographic Analysis of Ciliate Euplotes Octocarinatus Release Factor Erf1a

Zhang S., Zhou W., Ding Y., He X., Zhang H., Gao F., Heckmann K., Liang A & Rao Z.

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 P2₁ and the unit-cell parameters are a=90.4, b=107.9, c=114.8Å, b=94.2 ⁰ . There appear to be four eRF1a molecules in the asymmetric unit