Overexpression and Mechanistic Characterization of Blastula Protease 10, a Metalloprotease Involved in Sea Urchin Embryogenesis and Development

Abstract

Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1. Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated "tyrosine switch." It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared with the zinc form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes, which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloidrelated metalloenzyme involved in sea urchin embryogenesis.The astacin family of zinc-dependent endopeptidases is ubiquitously distributed across all phyla and part of the superfamily of metzincins (1). Approximately 30 members of the astacin family have been characterized at the protein level (2), such as meprins, bone morphogenetic protein-1 (BMP-1), 3 and tolloid, whereas several others have been identified through gene sequencing, including those in Caenorhabditis elegans (3). The signature of the sequence of the active site motif for this family of enzymes is HEXXHXXGXXH, where one Zn 2ϩ ion coordinates with the three histidines (boldface type), a tyrosine (Met-His/SerTyr in a loop region downstream from the coordinated His residues), and a water molecule (4). Most members of this family share common domain structures such as the pre-and proenzyme sequences located immediately N-terminal to the protease domain. Several members contain one or two copies of epidermal growth factor (EGF)-like domains and complement-like domains (Clr, Cls) near the C terminus (2). The shuffling of different domains in relation to the catalytic protease domain creates a variety of proteins with different structures and functions.Originally isolated and characterized as a developmentally regulated gene in sea urchin embryos (5, 6), the BP10 protein has remained uncharacterized. It shares sequence similarity with other members of the astacin family of enzymes (astacin itself being a crayfish digestive enzyme (4, ...

Defensome against Toxic Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

“…Previous reports have shown that hsp60 and hsp56 protein levels increase after heat shock, manganese and cadmium exposure in P. lividus embryos [3], [7], [18], [35]. Both hat and BP10 are early embryonic messengers, transiently expressed during the blastula stage [36]–[38]. A noteworthy observation from this study is the increased expression level of these proteases, when both genes are expressed.…”

Metal Complexes of a Multidentate Cyclophosphazene with Imidazole‐Containing Side Chains for Hydrolyses of Phosphoesters – Bimolecular vs. Intramolecular Dinuclear Pathway

“…Despite its high Lewis acidity, Cu II does not activate many Zn enzymes such as carboxypeptidase A and carbonic anhydrase 25. However, a few Zn hydrolases can be significantly activated by Cu II (BP10,26 serralysin,27 and astacin28). Presumably the more restricted tetragonally distorted Cu II coordination relative to the flexible Zn II coordination may account for the low activities in Cu II ‐substituted Zn enzymes.…”

Transcriptomic Insights into the Diversity and Evolution of Myxozoa (Cnidaria, Endocnidozoa) Toxin-like Proteins

“…Metalloproteases are important venom components of terrestrial animals [ 66 ]. They have also been detected in jellyfishes and sea urchins [ 67 , 68 , 69 ] with gelatinolytic, caseinolytic, and fibrinolytic activity [ 49 ]. The toxicity of CRISP in cnidarians is not fully understood, but it has been detected in anthozoans, scyphozoans, and hydrozoans [ 1 ].…”