The lysozyme of the starfish
Asterias rubens
A paradigmatic type
i
lysozyme
Sana Bachali
1
, Xavier Bailly
2
, Jacqueline Jolle
`
s
3
, Pierre Jolle
`
s
3,
* and Jean S. Deutsch
1
1
E
´
quipe De
´
veloppement et E
´
volution, UMR 7622 ‘Biologie du de
´
veloppement’, CNRS et Universite
´
P & M Curie, Paris, France;

the function of several conserved residues.
Keywords: cDNA; invertebrates; lysozyme; starfish; struc-
ture.
During recent years, interest in a new type of lysozyme, the
invertebrate-type (i-type), has been growing. In 1996 Jolle
`
s
et al. [1] published the N-terminal sequences of lysozymes
from two coastal bivalves belonging to the genus Mytilus
and of four deep-sea bivalves belonging to the genera
Bathymodiolus and Calyptogena. This lysozyme represented
a model for the digestion of bacteria by the deep-sea
bivalves [2]. A similar lysozyme was then described in other
bivalves, Tapes japonica [3], and Chlamys islandica [4,5].
These authors noticed the striking similarity between the
bivalve lysozyme and another protein, the so-called desta-
bilase identified in the medicinal leech Hirudo medicinalis
[6,7]. It was then determined that the leech destabilase also
has lysozyme activity [8,9].
In a previous work [10], we reported the cDNA
sequence of several bivalve lysozymes. We showed that,
in addition to bivalve lysozymes, homologous sequences
can be found in the genome of the nematode Caenorhab-
ditis elegans and that of the fly Drosophila melanogaster,as
well as expressed sequences tags from penaeid shrimps,
indicating that these species possess putative proteins akin
to the lysozyme i type. We performed a phylogenetic
analysis of all of these sequences together with those of the
more conventional lysozyme c type; the results suggested
that these two lysozymes originate from a common gene

s et al. [12], using iodoacetamide for alkylation. Diges-
tion by trypsin or carboxypeptidase (Worthington, Lake-
wood, NJ, USA)
1
or by Staphylococcus aureus V8 proteinase
(Miles) was performed for 18 h at 37 °Cin0.1
M
ammo-
nium bicarbonate with an enzyme/substrate ratio of 1 : 50.
Cyanogen bromide (Merck) cleavage was performed in
Correspondence to J. S. Deutsch, E
´
quipe De
´
veloppement et E
´
volution,
UMR 7622 ‘Biologie du de
´
veloppement’, CNRS et Universite
´
P&M
Curie, 9 quai St-Bernard, case 241, 75252 Paris cedex 05, France.
Fax: +33 14427 3253, Tel.: +33 14427 2576,
E-mail:
Note: The nucleotide sequence of the Asterias lysozyme i cDNA is
available in the GenBank database under accession number
AY390770.
*Present address: MNHN, Paris and Mine
´

`
s and Jolle
`
s[11].
This yielded too short a fragment to represent the complete
3¢ end of the lysozyme cDNA. Yet, its sequence showed a
clear homology with other lysozyme i sequences [10]. We
thought that this was due to inappropriate priming of the
oligo-dT primer. The specific (nondegenerated) primer AS3
(Table 1) was determined from this first sequence fragment.
Then a second PCR step was performed using AS3 and the
oligo-dT.
PCR were performed on cDNA in 20-lL reaction mix
containing 5¢- and oligo-dT primers at 20 and 4 pmol,
respectively, dNTP 10 m
M
and 5 U Qbiotaq DNA poly-
merase (Q-Biogene). PCR cycles were as follows: 3 min at
94 °C followed by 30–40 cycles of 1 min at 94 °C, 1 min at
56–59 °C (depending on the primers), 1 min at 72 °Cand
finally 10 min at 72 °C.
After amplification, the PCR products were analysed by
electrophoresis through 1% agarose gels and purified using
the Jetsorb Kit (Genomed). They were cloned in a
T-overhang vector derived from pBlueScript KS+ (Strata-
gene), prepared according to Holton [13]. Sequencing was
performed on both strands with the thermosequenase
fluorescent-labelled primer cycle sequencing kit and
7-deaza-dGTP (Amersham Pharmacia).
To expand the cDNA on its 5¢ side, the specific antisense

Results and discussion
Determination of the primary structure of the protein
The primary structure of the lysozyme from A. rubens was
determined by amino acid sequence analysis of the intact
carboxy-methylated protein and of constituent peptides
obtained through digests by trypsin, S. aureus V8 protein-
ase, carboxypeptidase and cyanogen bromide treatment (see
Material and methods). The results are summarized by the
sequence shown on Fig. 1.
cDNA cloning and sequencing
cDNA was prepared from soft tissues of a single A. rubens
specimen. As a starting point, we used degenerate primers
designed from the N-terminal sequence determined by Jolle
`
s
and Jolle
`
s [11] (Table 1). The complete cDNA sequence was
determined on PCR products after several rounds of 3¢ and
5¢ RACE/PCR (see Material and methods and Table 1).
Thus, the cDNA sequence was determined independently
of the biochemically determined protein sequence described
in the above paragraph.
The cDNA sequence agreed with the protein sequence
without ambiguity (Fig. 2) and allowed confirmation of the
data from the biochemical analysis in two cases when an
overlapping peptide was missing. The predicted ORF from
the cDNA is slightly longer than the biochemically deter-
mined protein sequence. Computer analysis (see Material
and methods) permitted us to postulate a signal peptide of

our previous work [10]. Comparison between the lysozyme
of the starfish, a deuterostome species, with the previously
known lysozymes i provides the opportunity to reveal
conserved residues over about 600 million years. Of about
120 amino acids, as many as 35 are identical, and 13 are
similar (Fig. 3). The starfish lysozyme is less rich in cysteines
than the protostome lysozymes i (10 vs. 13). Relative to the
other known i lysozymes, it presents a four-residue insertion
(residues 55–58 on Fig. 3). Comparison with the second
exon of the human lysozyme c that comprises the active site
reveals both similarities between the two types of lysozymes
and residues specific to the i-type (Fig. 3).
A
BLAST
search with the lysozyme sequence of A. rubens
in the sequence database of the National Center for
Biotechnology Information (NCBI) shows significant
sequence similarities with the destabilase of the medicinal
leech H. medicinalis [7], with the bivalve lysozyme sequences
determined in our previous work [10], with the lysozyme of
the bivalve Tapes japonica [3], with the so-called chlamysin
of Chlamys islandica [4,5], and also with a hypothetical
secreted protein of the nematode Caenorhabditis elegans and
with putative gene products retrieved from the genomes
of the fly Drosophila melanogaster and of the mosquito
Fig. 1. Chemically determined primary struc-
ture of the A. rubens lysozyme. Phenylalanine
112 is drawn in low case (f) because it was
ambiguous. >, Amino acid determined by
automated Edman degradation; <, amino

sapiens) is also aligned. Below are noted secondary structure elements: h marks a residue involved in an a-helix, b a residue involved in a beta-turn.
Residues conserved in all c-type lysozymes [21] are underlined. The two active acidic residues of the c-type lysozymes are boxed. Conserved residues
in all i-type lysozymes are in grey. Conserved cysteines are noted by s above the Asterias sequence.
Fig. 4. Hydrophobic cluster analysis. The primary sequence is represented on a roll mimicking a-helices. The primary sequence is drawn twice. A
dashed line follows one of these primary sequences. Prolines (P) and glycines (G) that break a-helices are represented as w and r, respectively. The
hydrophilic residues serines (S) and threonines (T) are represented by h. Residues that are distant on the primary sequence may appear close to each
other on this type of diagram, thus revealing hydrophobic clusters (boxed). (A) HCA plot of the Asterias lysozyme; hydrophobic residues conserved
in all lysozymes i are in grey. (B) Second exon of the human lysozyme c. Conserved hydrophobic residues between lysozymes i and c are in grey.
240 S. Bachali et al. (Eur. J. Biochem. 271) Ó FEBS 2003
have been lost in several deuterostome lineages. Compar-
ative genomics is developing rapidly. Complete genomes of
a greater panel of species will be soon available. This will
allow us to assess whether or not the lysozyme i gene has
been lost from the origin of the whole vertebrate or even the
whole chordate lineages. If this is true it this would fully
justify the name given of ‘invertebrate’ lysozyme.
Hydrophobic cluster analysis
Primary structure comparison between such distantly related
proteins as i-type and c-type lysozymes provides significant
yet insufficient data on functionally important residues. To
understand this issue further, we performed a hydrophobic
cluster analysis [14]. This analysis permits one to relate
residues that are not close to each other along the linear
sequence, but may come close under secondary structure,
forming hydrophobic clusters or pouches (Fig. 4A). The
N-terminal half of i lysozymes is homologous to the second
exon of vertebrate c-type lysozymes (Fig. 3 and [10]). Fig. 4B
shows the HCA plot of this part of the human lysozyme. A
number of hydrophobic residues overlap between this plot
and the corresponding part of the HCA plot of the Asterias

PSSM
E-value: 4.67 e-5)
2
.Inparticular,a
Fig. 5. Putative three-dimensional model of the Asterias lysozyme i. These figures were generated with the help of the
SWISS
-
PDBVIEWER
software. (A)
ApartoftheputativestructureoftheAsterias lysozyme, from residues L9 to L50 (according to numbering in Fig. 3). The side chains of E16 and
S34 that we postulate to be the active enzymatic residues (see text) are shown. (B) Homologous part of the human lysozyme from the model
deposited in the SwissProt data bank under the accession number 1IY3 (residues W28–K69, according to the sequence of the human lysozyme). The
side chains of E35 and D53 that are the known active residues in c-type lysozymes are shown.
Ó FEBS 2003 Lysozyme of Asterias rubens (Eur. J. Biochem. 271) 241
part of the three-dimensional structure of the Asterias
lysozyme (Fig. 5A) is very similar to the known structure of
the active site of the human c lysozyme (Fig. 5B). The
putative structure of the Mytilus lysozyme i is almost
identical (data not shown).
The critical glutamate (E) of lysozyme c active site is
conserved in lysozymes i. In contrast, the active aspartate
(D52 according to chicken numbering) is not conserved
(Fig. 3). In a previous paper we postulated that its role
could be played by another D residue [10]. The present
putative three-dimensional structure does not support this
hypothesis. On the other hand, the tertiary structure
supports the homology between the active D of lysozymes
c and a conserved serine (S), as proposed on the basis of
primary structure (Fig. 3). We determined the atomic
distances between the oxygen atom of this S and those of

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