Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family

Abstract

Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features.Keywords: lysozyme; invertebrate; purification; characterization; sequence.Lysozyme is a ubiquitous enzyme that is wildly distributed in the animal kingdom [1]. The biological function of this enzyme is believed to be self-defense from bacterial infection because it induces bacterial cell lysis by hydrolyzing b-1,4 linked glycosidic bond of the peptidoglycan on the bacterial cell wall [2].It is known that several types of lysozymes are distributed in the different parts of the animal kingdom. Goose-type and phage-type lysozymes are found in a part of avian [1] and bacterial phages [3], respectively. Other types of lysozymes are also seen in plants [4] and bacteria [5]. Chicken-type lysozyme is the most conventional one, being distributed in a large number of vertebrates [1] from fish to mammals and also found in insects like moths and flies [6]. Jolles and Jolles purified and partially sequenced another invertebrate lysozyme from a marine invertebrate, a starfish called Asterias rubens [7]. However, its N-terminal sequence was not similar to that of any known type of lysozyme including the chicken type lysozyme, implicating a novel type of lysozyme. Other invertebrate lysozymes had been purified and partially characterized [8±11], but the sequence data were not complete.To investigate the structure and function of invertebrate lysozyme, we purified lysozymes from three species of invertebrates: a marine bivalve, a marine conch, and an earthworm. We determined the N-terminal sequences of the three species and the complete sequence of the marine bivalve lysozyme. The biological function and evolutionary importance of this enzyme were discussed on the basis of the sequence and enzymatic properties. EXPERIMENTAL PROCEDURES Purification of bivalve lysozymeMarine bivalves (Tapes ...

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme*

“…As shown in Figure 5(A), the mass spectrum of the wild-type HEL (the molecular mass of HEL is 14309.4 or 14314.5) was unchanged after incubation with (NAG) 6 for 1 h at pH 3 and 408C, which was similar to the previous result. 8 While the mass spectrum of the N46E/D52S lysozyme showed a product with a molecular mass (15,116.4 Da) that was higher than the free enzyme mass (14,300.9 Da; theoretical mass: 14,300.1 Da).…”

“…4(A). Comparing with the HPLC profile of the equilibrated a-and b-anomers, hydrolysis of (NAG) 6 by wild type HEL and Vp lysozyme primarily gave the b-anomer of (NAG) 4 . 8,15 After hydrolysis of (NAG) 6 by N46D/D52S or N46E/ D52S lysozyme mutant for 15 min at 408C, the reaction mixture was injected onto an HPLC column.…”

“…[5][6][7] The i-type lysozyme is distinct from the other type lysozymes in the primary structure level. We recently elucidated the X-ray crystallographic structure of an invertebrate lysozyme from a bivalve, Venerupis philippinarum (Vp) (formerly Tapes japonica).…”

“…1). Furthermore, at pH 3.0, the (NAG) 6 hydrolysis of Vp lysozyme proceeded through a covalent glycolsyl intermediate by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. 8 Herein, we designed HEL mutants, Asn46Asp/ Asp52Ser (N46D/D52S) and Asn46Glu/Asp52Ser (N46E/D52S), in which catalytic residue replaced like Vp lysozyme and additionally one carbon atom appended to carboxylate group at 46th residue, respectively (Fig.…”

“…The mutant lysozymes had the sugar hydrolysis activities, which catalyzed NAG oligomer as similar to wild type HEL and Vp lysozymes. In particular, N46E/D52S mutant was found to accumulate a significant amount of the glycosyl adduct during the hydrolysis reaction with (NAG) 6 . This observation indicated that the catalysis of N46E/D52S lysozyme occurs through a covalent glycosyl intermediate like Vp lysozyme under low pH condition.…”