Lachesana tarabaevi, an expert in membrane-active toxins

In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (∼80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18–79 amino acid residues) comprise five major groups: repetitive polypeptide elements (Rpe), latarcins (Ltc), met-lysines (MLys), cyto-insectotoxins (CIT) and latartoxins (LtTx). Rpe are short (18 residues) amphiphilic molecules that are encoded by the same genes as antimicrobial peptides Ltc 4a and 4b. Isolation of Rpe confirms the validity of the iPQM (inverted processing quadruplet motif) proposed to mark the cleavage sites in spider toxin precursors that are processed into several mature chains. MLys (51 residues) present ‘idealized’ amphiphilicity when modelled in a helical wheel projection with sharply demarcated sectors of hydrophobic, cationic and anionic residues. Four families of CIT (61–79 residues) are the primary weapon of the spider, accounting for its venom toxicity. Toxins from the CIT 1 and 2 families have a modular structure consisting of two shorter Ltc-like peptides. We demonstrate that in CIT 1a, these two parts act in synergy when they are covalently linked. This finding supports the assumption that CIT have evolved through the joining of two shorter membrane-active peptides into one larger molecule.

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Design of Membrane Active Peptides with Regularly Repeating Glycine or D-amino Acid Residues

Understanding Biology Using Peptides ◽

10.1007/978-0-387-26575-9_276 ◽

2010 ◽

pp. 627-628

Author(s):

Jyothi Thundimadathil ◽

Roger W. Roeske ◽

Lili Guo

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Active Peptides ◽

Membrane Active Peptides

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The Primary Structure of Antithrombin-III

10.1055/s-0039-1680621 ◽

1977 ◽

Author(s):

T. E. Petersen ◽

G. Dudek-Wojciechowska ◽

L. Sottrup-Jensen ◽

S. Magnusson

Keyword(s):

Amino Acid ◽

Antithrombin Iii ◽

Factor Xa ◽

Amino Acid Residues ◽

Disulfide Bridges ◽

Single Chain ◽

Terminal Sequence ◽

Chymotryptic Peptide ◽

Sequence Region ◽

Bovine Factor

Human antithrombin-III is a single-chain glycoprotein with three disulfide bridges and four prosthetic glucosamine-based oligosaccharide groups. The disulfide bridges have been established. In four fragments of 208, 168, 3 and 46 amino acid residues, resp. 415 of the appr. 425 residues have been sequenced. The four oligosaccharide groups are attached to four Asn-residues within a sequence region of 95 residues. No extensive sequence homology with the trypsin inhibitors has been observed. One chymotryptic peptide was found to be a substrate for bovine factor Xa, cleaving the arginyl bond in the sequence -Ile-Val-Ala-Glu-Gly-Arg-Asp-. A second peptide is cleaved by thrombin. It is not clear whether these sites are inhibitor sites in the native molecule. Other possible candidates for inhibitor sites are a -Val-Leu-Ile-Leu-Pro-Lys-Pro- sequence (similar to the sequence 40-48 of hirudin, which also includes a -Pro-Lys-Pro- sequence) and also the C-terminal sequence -Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys.

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Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0220 ◽

2017 ◽

Vol 95 (6) ◽

pp. 634-643

Author(s):

Juliano Alves ◽

Miguel Garay-Malpartida ◽

João M. Occhiucci ◽

José E. Belizário

Keyword(s):

Amino Acid ◽

Large Subunit ◽

Small Subunit ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Wild Type ◽

Caspase 7 ◽

Caspase Family ◽

The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

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Localization of Ca2+-dependent conformational changes of calretinin by limited tryptic proteolysis

Biochemical Journal ◽

10.1042/bj3080607 ◽

1995 ◽

Vol 308 (2) ◽

pp. 607-612 ◽

Cited By ~ 24

Author(s):

J Kuźnicki ◽

T L Wang ◽

B M Martin ◽

L Winsky ◽

D M Jacobowitz

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Sequence Analysis ◽

Conformational Changes ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Amino Acid Sequence Analysis ◽

Sds Page ◽

Ef Hand ◽

Digestion Pattern

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

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The design of congener breaking sets to determine the function of amino acid residues in biologically active peptides

American Peptide Symposia - Peptides Frontiers of Peptide Science ◽

10.1007/0-306-46862-x_103 ◽

2005 ◽

pp. 253-254

Author(s):

James A. Burton ◽

Timothy B. Frigo ◽

Bore G. Raju

Keyword(s):

Amino Acid ◽

Biologically Active ◽

Amino Acid Residues ◽

Active Peptides ◽

Biologically Active Peptides

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Mode of cleavage of pig big endothelin-1 by chymotrypsin. Production and degradation of mature endothelin-1

Biochemical Journal ◽

10.1042/bj2700541 ◽

1990 ◽

Vol 270 (2) ◽

pp. 541-544 ◽

Cited By ~ 24

Author(s):

M Takaoka ◽

Y Miyata ◽

Y Takenobu ◽

R Ikegawa ◽

Y Matsumura ◽

...

Keyword(s):

Amino Acid ◽

Major Product ◽

Proteolytic Processing ◽

Intermediate Form ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Disulphide Bridge ◽

Endothelin 1 ◽

Hydrolysis Of

Pig endothelin-1 [ET-1-(1-21)] seems to be produced via proteolytic processing between Trp-21 and Val-22 of an intermediate form consisting of 39 amino acid residues, termed big ET-1-(1-39), by a chymotrypsin-like proteinase. We examined the chymotryptic-cleavage sites of big ET-1-(1-39) by reverse-phase h.p.l.c. and sequence analysis, and found that chymotrypsin cleaved initially the Tyr-31-Gly-32 bond of big ET-1-(1-39), followed by cleavage between Trp-21 and Val-22. Furthermore, chymotrypsin hydrolysed the generated ET-1-(1-21), producing a single major product that had the same amino acid sequence as ET-1-(1-21) with a cleavage between Tyr-13 and Phe-14. The disulphide bridge between Cys-1 and Cys-15 remained intact. These results indicate that the conversion of big ET-1-(1-39) into ET-1-(1-21) catalysed by chymotrypsin requires hydrolysis of the Tyr-31-Gly-32 bond before that of the Trp-21-Val-22 bond, an event followed by cleavage between Tyr-13 and Phe-14 within the loop of ET-1-(1-21). Thus a chymotrypsin-like proteinase might be involved not only in the production but also in the degradation of ET-1-(1-21) in vivo.

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Amino Acid Sequence Studies of Horseradish Peroxidase. II. Thermolytic Peptides

Canadian Journal of Biochemistry ◽

10.1139/o72-009 ◽

1972 ◽

Vol 50 (1) ◽

pp. 63-90 ◽

Cited By ~ 32

Author(s):

K. G. Welinder ◽

L. B. Smillie

Keyword(s):

Amino Acid ◽

Horseradish Peroxidase ◽

Amino Acid Sequence ◽

Cation Exchanger ◽

Paper Electrophoresis ◽

Tryptophan Residue ◽

Amino Acid Residues ◽

Disulfide Bridges ◽

Serine Residue ◽

C Terminus

Horseradish peroxidase (HRP) was digested with thermolysin. On fractionation on Sephadex G-25, Fine Chromobeads type P (Dowex 50 type resin) and by high-voltage paper electrophoresis, we isolated about 120 thermolytic peptides. Some experimentation on the composition of the pyridine acetate gradient, used for elution of the cation exchanger, is reported. All peptides were characterized with respect to amino acid composition, N-terminal residue, and pH 6.5 mobility. Unknown peptides or peptides not corresponding unambiguously to previously established tryptic sequences were subjected to dansyl-Edman analysis. Thermolytic peptides accounting for all tryptic sequences except a dipeptide and a tripeptide, and unique thermolytic sequences accounting for about 100 amino acid residues, were obtained. Nine convincing and several indicative overlaps were established for known tryptic sequences. The sequences around all four disulfide bridges, the three histidine residues, and the only tryptophan residue have been elucidated. Eight sites of carbohydrate attachment have been identified. For seven of these sites we have evidence for attachment to asparagine, and for six of the sites the carbohydrate-bound asparagine was found in the well-known sequences Asn–X–Ser/Thr. The remaining two sequences, though incomplete, are compatible with this pattern. Tentatively we suggest a pyrrolidone carboxyl N-terminal for HRP. The specificity of trypsin implicates a sequence found in two varieties, differing only by a C-terminal serine residue at the C-terminus of HRP. A discussion of the possible complications of the acidic heme extraction on the results obtained is included.

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Action of human liver cathepsin B on the oxidized insulin B chain

Biochemical Journal ◽

10.1042/bj2130467 ◽

1983 ◽

Vol 213 (2) ◽

pp. 467-471 ◽

Cited By ~ 34

Author(s):

M J McKay ◽

M K Offermann ◽

A J Barrett ◽

J S Bond

Keyword(s):

Amino Acid ◽

Cathepsin B ◽

Human Liver ◽

Degradation Products ◽

Cysteine Proteinase ◽

Cathepsin L ◽

Amino Acid Content ◽

Peptide Bond ◽

Cleavage Sites ◽

Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

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On the Primary Structure of Human Fibrinogen

10.1055/s-0039-1680394 ◽

1977 ◽

Author(s):

A. Henschen ◽

F. Lottspeich ◽

E. Töpfer-Petersen ◽

R. Warbinek

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Primary Structure ◽

Cyanogen Bromide ◽

Attachment Site ◽

Complete Sequence ◽

Cleavage Sites ◽

Amino Acid Residues ◽

Human Fibrinogen ◽

Β Chain

The aim of the present investigation is to elucidate the primary structure of human fibrinogen. Through the work of several laboratories including our own large parts of the structure are now known. Here will be reported the complete amino acid sequence of the γ-chain (409 residues). Furthermore, the carbohydrate linkage site in the β-chain and plasmin cleavage sites in the β- and γ-chains have been identified.The peptide chains were isolated by CM-cellulose chromatography following mercaptolysis and alkylation. The γ-chain was cleaved in a way to produce large fragments suitable for automatic sequencing, e. g. with cyanogen bromide or trypsin after citraconylation. The sequences of the isolated fragments allowed reconstruction of the complete sequence of the γ-chain.The carbohydrate linkage site in the β-chain could be isolated by affinity chromatography on concanavalin A-agarose following cleavage of the chain by trypsin or cyanogen bromide. The sequence of 21 amino acid residues around the carbohydrate attachment site was determined.The plasmin cleavage site giving rise to N-terminal glycine in the γ-chain D-fragment was identified. The amino acid sequence linking plasmic fragments E and D in the β-chain was determined.

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