Jón B. Bjarnason

Hemorrhagic metalloproteinases from snake venoms.

One of the more significant consequences of crotalid envenomation is hemorrhage. Over the past 50 years of investigation, it is clear that the primary factors responsible for hemorrhage are metalloproteinases present in the venom of these snakes. The biochemical basis for their activity is the proteolytic destruction of basement membrane and extracellular matrix surrounding capillaries and small vessels. These proteinase toxins may also interfere with coagulation, thus complementing loss of blood from the vasculature. Structural studies have shown that these proteinases are synthesized as zymogens and are processed at both the amino and carboxy termini to give the mature protein. The variety of hemorrhagic toxins found in snake venoms is due to the presence of structurally related proteins composed of various domains. The type of domains found in each toxin plays an important role in the hemorrhagic potency of the protein. Recently, structural homologs to the venom hemorrhagic metalloproteinases have been identified in several mammalian reproductive systems. The functional significance of the reproductive proteins is not clear, but in light of the presence of similar domains shared with the venom metalloproteinases, their basic biochemical activities may be similar but with very different consequences. This review discusses the history of hemorrhagic toxin research with emphasis on the Crotalus atrox proteinases. The structural similarities observed among the hemorrhagic toxins are outlined, and the structural relationships of the toxins to the mammalian reproductive proteins are described.

Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases

The proteolytic activity of four hemorrhagic metalloproteinases (Ht-a, c, d, and e) isolated from the venom of the Western diamondback rattlesnake (Crotalus atrox) was investigated using isolated extracellular matrix (ECM) proteins. We determined that all of the proteinases are capable of cleaving fibronectin, laminin, type IV collagen, nidogen (entactin), and gelatins. However, none of the proteinases were proteolytic against the interstitial collagen types I and III or type V collagen. With all of the substrates listed above Ht-c and Ht-d produced identical digestion patterns, as would be expected for these isoenzymes. With fibronectin, Ht-a produces a different ratio of products from Ht-c and Ht-d, while Ht-e produces a unique pattern of digestion. Ht-e and Ht-a produced nonidentical patterns with the laminin/nidogen preparation although some similarity was shared between them as well as with the Ht-c/d digestion pattern. Similar results were also observed for these proteinases with nidogen 150 as the substrate. The type IV collagen digestion patterns by Ht-e and Ht-a were similar to the pattern observed with Ht-c/d but differed by two bands. The digestion patterns of the three gelatins produced by the proteinases show differences between Ht-c and Ht-d when compared to Ht-e and Ht-a. This investigation clearly shows that several of the ECM proteins are efficiently digested by these toxins. The proteinases have some digestion sites in common but show differing specificities. In addition, the range of ECM proteins digested by these hemorrhagic proteinases is nearly identical to that demonstrated by the ECM proteinase stromelysin (MMP-3). From these data, and the knowledge of the roles these ECM proteins have in maintaining basement membrane structural/functional integrity, one can envision that the degradation of these ECM proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites.

Snake venom metalloproteinases: Structure, function and relationship to the ADAMs family of proteins

A large number of zinc metalloproteinases of varying mol. wts and biological functions has been isolated from crotalid and viperid venoms. Over the past few years, structural studies on these proteinases have suggested their organization into four classes, P-I to P-IV. These proteinases are synthesized in the venom gland as zymogens which are subsequently processed to the active form. The signal and pro-sequences of the proteins are highly conserved. Within the pro-domain lies a consensus sequence which probably functions in a manner similar to the cysteine switch in mammalian collagenases. The proteinase domain is represented by two forms: a two-disulfide and a three-disulfide structure. Crystallographic and modeling studies suggest that the two forms share very similar tertiary structures. The larger venom metalloproteinases (P-II, III and IV) have additional domains on the carboxy side of the proteinase domain. The additional domains that have been identified include disintegrin and disintegrin-like domains, a high-cysteine domain and a lectin-binding domain. It appears that these non-enzymatic domains function to modulate the biological properties of the proteinases. Recently, a family of homologues of the venom zinc metalloproteinases has been described from a variety of organisms including mammals, reptiles and invertebrates. This family of proteins has been termed the ADAMs, for A Disintegrin-like And Metalloproteinase-containing protein. They differ from the venom proteinases in that some of them may not have proteolytic activity. In addition to the domain structure described for the P-III class of venom proteins, the ADAMs have an epidermal growth factor-like domain, a transmembrane domain and a cytoplasmic domain. A description of venom metalloproteinase structure will be outlined in this review, along with the similarities and differences among the venom proteins and the ADAMs family of proteins.

Hemorrhagic Toxins from Snake Venoms

AbstractOne of the more dramatic consequences of envenomation by crotalid and viperid snakes is the occurrence of hemorrage. In cases where the envenomation is less severe, the hemorrhagic is generally observed to be localized at the site of the bite. However, hemorrhage can be found disseminated through a substantial area of the involved extremity. In cases where the envenomation is severe, bleeding in organs such as heart, lungs, kidneys and brain may also occur. From the biochemical investigations on these toxins over the past 30 years, the nature of the venom toxins and their mechanism of activity are now becoming clear. Virtually all of the hemorrhagic toxins isolated and characterized thus far have been determined to be metalloproteinases. In this review we discuss the history of the isolation and characterization of these toxins in an attempt to clarify some of the confusion surrounding these toxins and their biochemical activities. We also survey the data available on the natural and synthetic inh...

Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.

Properties of elastase from Atlantic cod a cold-adapted proteinase

An intestinal elastase was purified from Atlantic cod (Gadus morhua) of apparent molecular mass 24.8 kDa as determined by SDS-PAGE and with isoelectric point above pI 9.3. Heat stability and stability towards acidic pH was reduced in the cod enzyme as compared with porcine intestinal elastase. N-terminal amino-acid sequence analysis of cod elastase showed considerable similarity with porcine elastase. The cod enzyme was less sensitive to phenylmethylsulfonyl fluoride inhibition than porcine elastase, but sensitivity towards other inhibitors was similar. Kinetic properties were examined using the substrate Suc-Ala-Ala-Ala-p-nitroanilide and the cod enzyme was found to have more than 2-times turnover rate (kcat) as compared with the porcine enzyme, and slightly higher Km values. Thus, the catalytic efficiency (kcat/Km) of Atlantic cod elastase was about 2-times higher than observed with porcine elastase, which indicates an adaptive response towards the low temperature environmental in which the cod lives. Substrate specificity was studied by digestion of oxidized B-chain of insulin and by using synthetic substrates. Digestion was most rapid at the carbonyl side of alanine residues, but also occurred at valine and leucine residues.

Catalytic properties and chemical composition of pepsins from Atlantic cod (Gadus morhua)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.

Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua).

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (+/- 0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C.

Identification of the Cleavage Sites by a Hemorrhagic Metalloproteinase in Type IV Collagen

Type IV collagen, solubilized from Engelbreth-Holm-Swarm (EHS) tumor basement membranes is digested by a hemorrhagic metalloproteinase, Ht-e, isolated from the crude venom of the Western Diamondback rattlesnake, Crotalus atrox. The major proteolytic products have Mr 141,000, 132,000, 87,000, 71,000, 33,000 and approximately 18,000 as estimated by SDS-gel electrophoresis of pepsinized type IV collagen fragments. Sequence analysis of the digestion products reveal that the Mr 141,000, 71,000 and approximately 18,000 band are derived from the alpha 1(IV) chains and the Mr 132,000, 87,000 and 33,000 bands are derived from the alpha 2(IV) chain. The products are stable over 72-hour incubation periods. The cleavage sites on the alpha 1(IV) and alpha 2(IV) chains are not identical. The alpha 1(IV) chains are cleaved in a pepsin susceptible triplet interruption region of the triple helix at position Ala258-Gln259. The alpha 2(IV) chain is cleaved in the triple helical region near the NC2 domain at the Gly191-Leu192 peptide bond. Isolated hexameric NC1 globular domains of type IV collagen are not digested by Ht-e. The present study demonstrates that the venom hemorrhagic metalloproteinase Ht-e has type IV collagenolytic activity. The triple helix of the type IV collagen molecule is cleaved in a region located immediately carboxyl to the flexible NC2 domain. The degradation by Ht-e of type IV collagen, a major component of basement membranes which forms the scaffold of this extracellular structure, may account in part for the hemorrhagic activity of this toxin.

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake (Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.