Evidence that the platinum-reactive methionyl residue of the alpha 2-macroglobulin receptor recognition site is not in the carboxyl-terminal receptor binding domain

Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.     

Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen).

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.     

Comparative genomic study of arachnid immune systems indicates loss of beta‐1,3‐glucanase‐related proteins and the immune deficiency pathway

Analyses of arthropod genomes have shown that the genes in the different innate humoral immune responses are conserved. These genes encode proteins that are involved in immune signalling pathways that recognize pathogens and activate immune responses. These immune responses include phagocytosis, encapsulation of the pathogen and production of effector molecules for pathogen elimination. So far, most studies have focused on insects leaving other major arthropod groups largely unexplored. Here, we annotate the immune‐related genes of six arachnid genomes and present evidence for a conserved pattern of some immune genes, but also evolutionary changes in the arachnid immune system. Specifically, our results suggest that the family of recognition molecules of beta‐1,3‐glucanase‐related proteins (βGRPs) and the genes from the immune deficiency (IMD) signalling pathway have been lost in a common ancestor of arachnids. These findings are consistent with previous work suggesting that the humoral immune effector proteins are constitutively produced in arachnids in contrast to insects, where these have to be induced. Further functional studies are needed to verify this. We further show that the full haemolymph clotting cascade found in the horseshoe crab is retrieved in most arachnid genomes. Tetranychus lacks at least one major component, although it is possible that this cascade could still function through recruitment of a different protein. The gel‐forming protein in horseshoe crabs, coagulogen, was not recovered in any of the arachnid genomes; however, it is possible that the arachnid clot consists of a related protein, spätzle, that is present in all of the genomes.     

Genomic and exoproteomic analyses of cold‐ and alkaline‐adapted bacteria reveal an abundance of secreted subtilisin‐like proteases

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography–tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin‐like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20°C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

Evidence for a two-step mechanism involved in the formation of covalent HC x TSG-6 complexes

IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2.     

Extracellular superoxide dismutase exists as an octamer

Human extracellular superoxide dismutase (EC-SOD) is involved in the defence against oxidative stress induced by the superoxide radical. The protein is a homotetramer stabilised by hydrophobic interactions within the N-terminal region. During the purification of EC-SOD from human aorta, we noticed that material with high affinity for heparin-Sepharose formed not only a tetramer but also an octamer. Analysis of the thermodynamic stability of the octamer suggested that the C-terminal region is involved in formation of the quaternary structure. In addition, we show that the octamer is composed of both aEC-SOD and iEC-SOD folding variants. The presence of the EC-SOD octamer with high affinity may represent a way to influence the local concentration of EC-SOD to protect tissues specifically sensitive to oxidative damage.     

Divorcing folding from function: How acylation affects the membrane-perturbing properties of an antimicrobial peptide

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an α-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8–C16 alkyl chains and the inclusion of anionic lipids both increase Nc's ability to form α-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of α-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of α-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing “plugging” effect.