Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

Analytical, physiologic, and clinical validation of a radioimmunoassay for measurement of procollagen type III amino terminal propeptide in serum and bronchoalveolar lavage fluid obtained from dogs.

OBJECTIVE To validate a radioimmunoassay for measurement of procollagen type III amino terminal propeptide (PIIINP) concentrations in canine serum and bronchoalveolar lavage fluid (BALF) and investigate the effects of physiologic and pathologic conditions on PIIINP concentrations. SAMPLE POPULATION Sera from healthy adult (n = 70) and growing dogs (20) and dogs with chronic renal failure (CRF; 10), cardiomyopathy (CMP; 12), or degenerative valve disease (DVD; 26); and sera and BALF from dogs with chronic bronchopneumopathy (CBP; 15) and healthy control dogs (10 growing and 9 adult dogs). PROCEDURE A radioimmunoassay was validated, and a reference range for serum PIIINP (S-PIIINP) concentration was established. Effects of growth, age, sex, weight, CRF, and heart failure on S-PIIINP concentration were analyzed. In CBP-affected dogs, S-PIIINP and BALF-PIIINP concentrations were evaluated. RESULTS The radioimmunoassay had good sensitivity, linearity, precision, and reproducibility and reasonable accuracy for measurement of S-PIIINP and BALF-PIIINP concentrations. The S-PIIINP concentration reference range in adult dogs was 8.86 to 11.48 mug/L. Serum PIIINP concentration correlated with weight and age. Growing dogs had significantly higher S-PIIINP concentrations than adults, but concentrations in CRF-, CMP-, DVD-, or CBP-affected dogs were not significantly different from control values. Mean BALF-PIIINP concentration was significantly higher in CBP-affected dogs than in healthy adults. CONCLUSIONS AND CLINICAL RELEVANCE In dogs, renal or cardiac disease or CBP did not significantly affect S-PIIINP concentration; dogs with CBP had high BALF-PIIINP concentrations. Data suggest that the use of PIIINP as a marker of pathologic fibrosis might be limited in growing dogs.

Platelet glycoprotein Ia* is the processed form of multimerin--isolation and determination of N-terminal sequences of stored and released forms

Glycoprotein Ia* (GPIa*), a very high molecular mass, platelet alpha-granule protein consisting of 167 kDa subunits disulphide-linked in a multimeric structure, was first described by Bienz and Clemetson in 1989 (J. Biol. Chem. 264, 507-514). In 1991 Hayward et al. (J. Biol. Chem. 266, 7114-7120) independently identified a platelet protein with multimeric structure. Despite strong similarities to GPIa* they concluded that it was a novel multimeric protein and named it first p-155 and later, multimerin. Multimerin has also been found in endothelial cells and has been cloned recently from an endothelial cell cDNA library. This has made it possible for us to clarify the relationship between GPIa* and multimerin. GPIa* was isolated from platelet releasate and the N-terminal sequence of 167 kDa and 155 kDa subunit species were determined. The N-terminal 15 amino acids of GPIa* were identical to the deduced amino acids 184-198 of endothelial multimerin. The N-terminal sequence of the 155 kDa protein was identical to the deduced amino acids 318-326 of multimerin. Thus, platelet GPIa* (167 kDa) is the main processed form of multimerin stored in platelet alpha-granules. The GPIa*/processed multimerin (167 kDa) still contains an RGDS sequence near its N-terminus as well as an EGF domain which may be involved in binding to the platelet surface after release. This sequence and domain are cleaved off in the p-155 form, described earlier as platelet multimerin, which is probably formed after release from alpha-granules.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.

Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

STRUCTURE OF CUPIENNIUS SALEI VENOM HYALURONIDASE Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. FUNCTION OF VENOM HYALURONIDASES Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well the current phylogenetic idea on a more isolated position of these families and can perhaps be explained by specialized prey catching techniques.

Structures of SAS-6 suggest its organization in centrioles

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.