Biol. Chetn. Hoppe-Seyler Vol. 373, pp. 381-385, July 1992
A New Family of Proteinases is Defined by Several Snake Venom Metalloproteinases LAWRENCE A. HiTEa, JAY w. Foxa and JON B. BjARNASONb a b
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A. Science Institute, University of Iceland, Dunhagi 3, IS-107 Reykjavik, Iceland
Summary Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms. Introduction Crotalid snake venoms and their activities have been the object of much investigation for decades. A prominent feature of crotalid snake venoms is that they contain an abundance of proteolytic enzymes. While much has been learned about the physiological effects of these venoms, the enzymes responsible for these effects have often proven to be difficult to purify and characterize. One example is the group of venom components which cause hemorrhage. Early studies on snake venom hemorrhagic toxins were often inconclusive and misleading, mainly due to uncertainties about the purity of isolated hemorrhagic toxins as well as primitive enzyme assays. Eventually, thorough investigations proved them to be zinc metalloproteinases of a unique nature [1]. More recent studies have shown these proteinases to be capable of hydrolyzing basement membrane components, including laminin, nidogen, fibronectin and collagen type IV [2,3]. Recently, the protein sequences
Enzymes: Hemorrhagic toxin a or atrolysin a (EC 3.4.24.1); Hemorrhagic toxin d or atrolysin c/d (EC 3.4.24.42); Hemorrhagic toxin e or atrolysin e (EC 3.4.24.44); Hemorrhagic protein IB or trimerelysin I (EC 3.4.24.52); Hemorrhagic protein 2a or trimerelysin II (EC 3.4.24.53); Thermolysin (EC 3.4.24.27); Hemorrhagic factor-II (LHF-II) from L. muta muta venom; Hemorrhagic toxin-2 (HT-2) from C. ruber ruber venom; H2-Proteinase from T.flavoviridis venom.
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382
L.A. Hite, J.W. Fox and J.B. Bjarnason
Vol. 373 (1992)
for five of the hemorrhagic toxins, Ht-d (hemorrhagic toxin d or atrolysin c/d) from Crotalus atrox venom [4], HR2a (hemorrhagic protein 2a or trimerelysin II) and HR1B (hemorrhagic protein IB or trimerelysin I) from Turneresurus flavoviridis venom [5,6], LHF-II (hemorrhagic factor-II) from Lachesis muta muta venom [7] and HT-2 (hemorrhagic toxin-2) from Crotalus ruber ruber venom [8], have been determined using Edman degradation. The sequence of a nonhemorrhagic metalloproteinase, H2-Proteinase, from T. flavoviridis venom has also been determined [9]. Furthermore, we have now completed the cDNA sequence of the hemorrhagic metalloproteinase Ht-e (hemorrhagic toxin e or atrolysin e, paper in press) from Crotalus atrox venom. Unexpectedly, this cDNA sequence shows strong similarity to the cDNA for the disintegrin trigramin from Trimeresurus gramineus venom [10]. Furthermore, there is evidence that this trigramin clone also encodes a zinc metalloproteinase. This brings the total number of venom metalloproteinase s for which the total sequence is available to eight and we may now draw some conclusins as to their interrelationship. Results and Discussion Figure 1 shows a comparison of the eight snake venom metalloproteinases for which sequence data is available. While these proteinases are quite similar to one another they bear no extended similarity to any other know proteins except the disintegrins (which are also snake venom components). They all share the consensus zinc-binding site first described for thermolysin [11] H-E-X-X-H, as well as the extended sequence first noted by Murphy et al. [12], (H-E-X-X-H)-X-X-G-X-X-H, which is also seen in the matrix metalloproteinases. There is actually an extended consensus sequence for the venom metalloproteinases, V-X-M-X-(H-E-X-G-H)-N-L-G-X-X-H-D. A computer analysis using the method of Gamier and Robson [13] predicts that this extended consensus lies at the end of an alpha helix of approximately 18 to 20 residues, ending in a beta turn. This is consistent with the data from the X-ray crystal structure for thermolysin [14]. Thus, it is evident that all of these venom metalloproteinases have a similar zinc-binding region. Another point of interest is the strong similarity of the nonhemorrhagic H2-Proteinase to the hemorrhagic proteinases. It has been hypothesized that the hemorrhagic proteinases would be distinctly different from their nonhemorrhagic counterparts. This does not appear to be the case as far as their sequences are concerned, and in some instances H2-Proteinase is actually more similar to some of the hemorrhagic proteinases than they are to each other (most notably H2-Proteinase is 57.3% identical to Ht-e, whereas Ht-e is only 54,8% identical to Ht-d, a hemorrhagic proteinase from the venom of the same snake). However, important differences have been noted for the hemorrhagic proteinases from Crotalus atrox venom in terms of their proteolytic activities. A correlation has been observed for the abilities of these hemorrhagic proteinases to induce hemorrhage and their ability to digest basement membrane in the form of matrigel [15]. Furthermore, the digestion patterns for isolated basement components, such as collagen type IV, laminin, nidogen and fibronectin, by these hemorrhagic proteinases is clearly different [2,3,16], We suggest, from the available sequence data, that the region likely to be important to the ability these proteinases to produce hemorrhage can be narrowed down to amino Brought to you by | Purdue University Libraries Authenticated Download Date | 6/4/15 11:09 PM
Vol. 373 (1992)
Ht-e r.£«min«if HR1B HR2a Η,-Prote iase Ht-d LHF-n HT-2
Snake Venom Metalloproteinases
383
1 10 20 30 40 50 N P E H Q R Y V E L F I V V D H G M Y T K Y N G D S D K I R Q R V H Q M V N I MKE S YT YMY I D F Q Q R F P Q R Y I K L G I F V D H G M Y T K Y S G N S ER I T K R V H Q M I N N I N M M C R A L N I V
A New Family of Proteinases is Defined by Several Snake Venom Metalloproteinases LAWRENCE A. HiTEa, JAY w. Foxa and JON B. BjARNASONb a b
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A. Science Institute, University of Iceland, Dunhagi 3, IS-107 Reykjavik, Iceland
Summary Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms. Introduction Crotalid snake venoms and their activities have been the object of much investigation for decades. A prominent feature of crotalid snake venoms is that they contain an abundance of proteolytic enzymes. While much has been learned about the physiological effects of these venoms, the enzymes responsible for these effects have often proven to be difficult to purify and characterize. One example is the group of venom components which cause hemorrhage. Early studies on snake venom hemorrhagic toxins were often inconclusive and misleading, mainly due to uncertainties about the purity of isolated hemorrhagic toxins as well as primitive enzyme assays. Eventually, thorough investigations proved them to be zinc metalloproteinases of a unique nature [1]. More recent studies have shown these proteinases to be capable of hydrolyzing basement membrane components, including laminin, nidogen, fibronectin and collagen type IV [2,3]. Recently, the protein sequences
Enzymes: Hemorrhagic toxin a or atrolysin a (EC 3.4.24.1); Hemorrhagic toxin d or atrolysin c/d (EC 3.4.24.42); Hemorrhagic toxin e or atrolysin e (EC 3.4.24.44); Hemorrhagic protein IB or trimerelysin I (EC 3.4.24.52); Hemorrhagic protein 2a or trimerelysin II (EC 3.4.24.53); Thermolysin (EC 3.4.24.27); Hemorrhagic factor-II (LHF-II) from L. muta muta venom; Hemorrhagic toxin-2 (HT-2) from C. ruber ruber venom; H2-Proteinase from T.flavoviridis venom.
Brought to you by | Purdue University Libraries Copyright © by Walter Authenticated de Gruyter & Co · Berlin · New York
Download Date | 6/4/15 11:09 PM
382
L.A. Hite, J.W. Fox and J.B. Bjarnason
Vol. 373 (1992)
for five of the hemorrhagic toxins, Ht-d (hemorrhagic toxin d or atrolysin c/d) from Crotalus atrox venom [4], HR2a (hemorrhagic protein 2a or trimerelysin II) and HR1B (hemorrhagic protein IB or trimerelysin I) from Turneresurus flavoviridis venom [5,6], LHF-II (hemorrhagic factor-II) from Lachesis muta muta venom [7] and HT-2 (hemorrhagic toxin-2) from Crotalus ruber ruber venom [8], have been determined using Edman degradation. The sequence of a nonhemorrhagic metalloproteinase, H2-Proteinase, from T. flavoviridis venom has also been determined [9]. Furthermore, we have now completed the cDNA sequence of the hemorrhagic metalloproteinase Ht-e (hemorrhagic toxin e or atrolysin e, paper in press) from Crotalus atrox venom. Unexpectedly, this cDNA sequence shows strong similarity to the cDNA for the disintegrin trigramin from Trimeresurus gramineus venom [10]. Furthermore, there is evidence that this trigramin clone also encodes a zinc metalloproteinase. This brings the total number of venom metalloproteinase s for which the total sequence is available to eight and we may now draw some conclusins as to their interrelationship. Results and Discussion Figure 1 shows a comparison of the eight snake venom metalloproteinases for which sequence data is available. While these proteinases are quite similar to one another they bear no extended similarity to any other know proteins except the disintegrins (which are also snake venom components). They all share the consensus zinc-binding site first described for thermolysin [11] H-E-X-X-H, as well as the extended sequence first noted by Murphy et al. [12], (H-E-X-X-H)-X-X-G-X-X-H, which is also seen in the matrix metalloproteinases. There is actually an extended consensus sequence for the venom metalloproteinases, V-X-M-X-(H-E-X-G-H)-N-L-G-X-X-H-D. A computer analysis using the method of Gamier and Robson [13] predicts that this extended consensus lies at the end of an alpha helix of approximately 18 to 20 residues, ending in a beta turn. This is consistent with the data from the X-ray crystal structure for thermolysin [14]. Thus, it is evident that all of these venom metalloproteinases have a similar zinc-binding region. Another point of interest is the strong similarity of the nonhemorrhagic H2-Proteinase to the hemorrhagic proteinases. It has been hypothesized that the hemorrhagic proteinases would be distinctly different from their nonhemorrhagic counterparts. This does not appear to be the case as far as their sequences are concerned, and in some instances H2-Proteinase is actually more similar to some of the hemorrhagic proteinases than they are to each other (most notably H2-Proteinase is 57.3% identical to Ht-e, whereas Ht-e is only 54,8% identical to Ht-d, a hemorrhagic proteinase from the venom of the same snake). However, important differences have been noted for the hemorrhagic proteinases from Crotalus atrox venom in terms of their proteolytic activities. A correlation has been observed for the abilities of these hemorrhagic proteinases to induce hemorrhage and their ability to digest basement membrane in the form of matrigel [15]. Furthermore, the digestion patterns for isolated basement components, such as collagen type IV, laminin, nidogen and fibronectin, by these hemorrhagic proteinases is clearly different [2,3,16], We suggest, from the available sequence data, that the region likely to be important to the ability these proteinases to produce hemorrhage can be narrowed down to amino Brought to you by | Purdue University Libraries Authenticated Download Date | 6/4/15 11:09 PM
Vol. 373 (1992)
Ht-e r.£«min«if HR1B HR2a Η,-Prote iase Ht-d LHF-n HT-2
Snake Venom Metalloproteinases
383
1 10 20 30 40 50 N P E H Q R Y V E L F I V V D H G M Y T K Y N G D S D K I R Q R V H Q M V N I MKE S YT YMY I D F Q Q R F P Q R Y I K L G I F V D H G M Y T K Y S G N S ER I T K R V H Q M I N N I N M M C R A L N I V
