The cytotoxin of Pseudomonas aeruginosa:Cytotoxicity requires proteolytic activation

The primary structure of a cytotoxin from Pseudomonas aeruginosa was determined by sequencing of the structural gene. The cytotoxin (31,700 Mr) lacks an N-terminal signal sequence for bacterial secretion but contains a pentapeptide consensus sequence commonly found in prokaryotic proteins which function in a TonB-dependent manner. The cytotoxin gene has a [G + C]-content of 53.8% which is considerably lower than generally observed for genes from Pseudomonas aeruginosa. The cytotoxin gene was exclusively detected in strain 158 but not in three other clinical isolates, as determined by Southern and Northern hybridization. The latter technique revealed that the toxin is translated from monocistronic mRNA. The promoter of the cytotoxin is inactive in Escherichia coli. Upon site-directed modification of the 5'-noncoding region by the polymerase chain reaction the gene was expressed under control of the trc-promoter. The gene product obtained in Escherichia coli was nontoxic. Toxicity was induced by subsequent treatment with trypsin. [35S]methionine-labeled cytotoxin with high specific radioactivity was obtained by in vitro transcription/translation. Like [125I] labeled material from Pseudomonas aeruginosa this polypeptide bound to membrane preparations from Ehrlich ascites cells, as evidenced by sedimentation through a sucrose gradient at neutral pH

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase

Assignment of disulphide bonds in human platelet GPIIIa. A disulphide pattern for the β-subunits of the integrin family

9 pags, 7 figs, 2 tabsIntegrins are cell-surface heterodimers formed by the association of one α- and one β-subunit. Glycoprotein IIIa (GPIIIa or β3 subunit) is the common β-subunit of the β3 subfamily of integrins, which, when associated with glycoprotein IIb (GPIIb), constitutes the receptor for fibrinogen and other adhesive proteins at the platelet surface (the GPIIb-IIIa complex) and, when associated with the α(v) subunit, constitutes the vitronectin receptor present in several cell types. Protein chemical analysis of GPIIIa allows us to define the following structural domains: the cysteine-rich and proteinase-resistant N-terminal domain (GPIIIa 1-62); the adhesive-protein-binding domain (GPIIIa 101-422); the cysteine-rich and proteinase-resistant core (GPIIIa 423-622); and the C-termina1 domain comprising an extracellular subdomain (GPIIIa 623-692), a transmembrane subdomain (GPIIIa 693-721), and a cytoplasmic subdomain (GPIIIa 722-762). We also assign unambiguously the disulphide bonds within the N-terminal, the fibrinogen-binding and the C-terminal domains, and the two long-range disulphide bonds which join the N-terminus to the proteinase-resistant core (Cys5-Cys435) and the fibrinogen-binding domain to the extracellular side of the C-terminal domain (Cys406-Cys655). In addition, we propose three alternative models for the arrangement of the disulphide bonds within the core and of the disulphide bonds joining the core to the extracellular side of the C-terminal domain, consistent with our experimental findings, favouring temporarily that which imposes less steric hindrance for the formation of these disulphide bonds. On the basis of this information and on the highly conserved overall structure observed in the β-subunits of the integrin family known so far, except in β4, we propose to extend the cysteine-pairing pattern and the structural domains outlined here for GPIIIa to all the β-subunits of the integrin family.This workwas supported by the Secretaria de Estado para Universidades e Investigación (ID 87077 SEUI; PM-022 SEUI) and an Accion Integrada Hispano Germana (1989-1990 43A)

Four crystal forms of a Bence-Jones protein

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases