Moleculat Matine BioJogy and BiolechnoJogy (1997) 6(3), 228-237

Amylase in Pecten maximus (Mollusca, bivalves): protein and

cDNA characterization; quantification of the expression in the
digestive gland

S. Le Moine
Ifremer, COB, BP 70, 29280, Plouzan, Brest. France
D. Sellos
Laboratoire de Bialogie Marine du Musum Nalianal
d'Histaire NaturelJe el du Collge de France, BP 225,
29182, Coticarneau, France
J. Moal*
lfremer, COB, BP 70, 29280, Plouzan,

Bresl, France

J.Y. Daniel
lfremer. COB, BP 70, 29280, Plouzon, Bresl, France
F. San Juan Serrano
Departamento de Biologia Fundamental, Facultad de
Ciencas, Universidad de Vigo, Aptdo 784, 36200 Vigo,
Spain
J.F. Sarnain
Ifremer, COB, BP 70, 29280, Plouzan,

Brest, France

A. Van Wormhoudt
Labotatoire de Biologie Matitie du Musum
ational
d'Histoire
oturelle et du Collge de France, BP 225,
29182, Concarneau, France
Abstraet
The digestive enzyme o-amylase in Pecten maximus has been purified frorn the digestive gland,
where it is present as two isoforms. In order to
gain information on its structure and regulation, a
digestive gland cD A library, constructed
in A
phage Zap II (Stratagene, La [olla, Calif., U.S.A.),
was screened with a shrimp o-amylase cD A probe.
Only 0.02% of the clones were positive, and the
longest clone, having a ize of 1700 bp and identical
to that of the mRNA. was fuUy sequenced. It contains the complete cDNA coding frame for one of
the amylase isoforms of P maximus. The deduced
protein sequence is 508 amino acids long, with a
putative 18 arnino acid, highly hydrophobic
signal

'Correspondenee
should be ent lo thi author.
1997 Blaekwell Seienee, Inc.

C>

228

peptide and a mature enzyme of 489 residues. The

molecular weight corresponds to 54,500 Da and the
calculated isoelectric point is 6.76. Location of conserved equences confirms the high level of similarity with the other members of the family (EMBL
Accession No. X99729).

Introduetion
In mollusk bivalves, digestion is seen as a complex
combination of intracellular and extracellular processes. Good relationships
between digestive enzymes and assimilation
rates were described in
scallop larvae and mussels (Samain et al., 1991;
Bayn , 1976). In the field as weU as in controlled
conditions in hatcheres, individual growth differences in a population
may be partially related to
different capacities to digest and assimilate ingested
food by differential
expression
of digestive enzymcs. In mollusks, the relations of enzyme level
with growth are not weU documented,
but a seasonal survey showed that instantaneous
growth of
Crassostrea gigas in the field was related to digestive enzyme activity (data not published). Paynter
and Chen (1991) also described a growth enhancement in C. virginica juveniles after short irnmersions
in biosynthetic
trout growth hormone and attributed this favorable effect to a better assimilation
rate in a deficient nutritional environment.
Many reports concern the adaptive responses of
pancreatic enzymes in res pon se to food substrates
and hormones in vertebrates, but very few concern
invertebrate
and e pecially mollusks. In rats, Desnuelle et al. (1962) and Dagorn and Lahaie (1981)
ha ve shown a direct relation between level of enzyrnes and their respective nutritional substrates.
For carbohydrates, this relation is well documented
and is under
hormonal
control
(Dagorn and
Mongeau. 1977). Insulin is the main hormone involved in rat arnylase regulation (Korc et al., 1981).
A wide range of carbohydrases
are expressed in
the digestive gland of mollusks (Stark and Walker,
1983), but their control is not yet elucidated. Amylas e activity appears in the digestive diverticula and

Amylase in Peclen maximus

stomach (Henry et al., 1993), and is included in the

crystalline stylc during the digestive proccss. Amylase activity was studied in the oystcr Ostrea edulis
(Mathers, 1973) and Pecten maximus
( tark and
Walker, 1983). The activity in creas e five hours after
foodsupplyin
G.gigas(Boucaud-Camou
eta1., 1983).
Despite the importance ofmarine mollusks, however, little is known about the structure of o-amylases (0.-1,4 glucan-4-gluconohydrolase,
EC 3.2.1.1).
In general, a amylase catalyzes the hydrolysis of
a-O (1,4) glucosidic
bonds in star h componcnts,
glycogen, and related oligosaccharides.
In scallop ,
the level of this enzyme is modified by phytoplanktonic composition,
ingestion levels, and developmental stages (Samain et al., 1991).
Since the first primary structure deduced from
rat pancreatic cD A (Rutter, 1980; Pictet et al.,
1981), reported sequences have been mainly for prokaryotic arnylases and some vertebrate enzyme including porcine, rat, and human.ln contrast, tudie
of o-amylase from invertebrates remain limited, and
only fruit fly and mosquito amylase sequences have
been determined (Boer and Hickey, 1986; Grossman
and [ames, 1993).
In this article we report for the first time the
purification, characterization.
and molecular cloning of o-amylase from Pecten maxitnus, which is
an important mollusk of commercial inter st. Th
structure of the encoded pro te in is analyzod and
com pared with the biochemical features oflhe purified enzyme. The expression of this enzymo in the
digestive gland, calculated either from sp cific activities or from the numbcr of positive clones in the
cD A library, is compared.

Results
Amylose

purificotion

The specific acti vity of arn lase in the crude extracts

of Pecten moximus digestive gland was 0.09 units/
mg protein. After purification on modified starch,
the specific activity was increased to 19 units/mg
protein, equivalent to a purification
of 200 and a
yield of 25% (Table 1). The purity of the enzyme
preparation wa examin d b poi acrylamide gel
electrophoresis under native and denaturing con ditions. The electrophoretic
pattern in native con ditions showed two protein bands, both exhibiting
amylase activity (Figure 1). On SDS electrophore
is
gel, only one protein fraction was identified (Figure
2). By motility compari on, the mol cular w ight
was estimated at 60 kDa (Figure 2). After dialysis

229

and ultrafiltration
the spccific activity was greatly
increasod, to 140 units/mg protein, leading to an
estimation of 0.065% of onzyme in the crude extract, assuming that the same activities were mea sured in crude extract and after purification. This
hypothesis will be discussed below.
o data were
obtained on amino acid sequence, the amino-terminus not being accessible for Edman degradation
(data not shown).
Cotnplementary
onalysis

D A library screeriing and cDNA

Only 0.02% clones from the cD A library were

positive. The longest positive clone, containing a
1700-bp insert, was characterized.
The complete
nucleotido sequence (Figure 3) reveals a single open
rcading frame of 1645 bp, without the poly(A) tail.
Th ATG tart odon was found 4 bp from the 5'
end of the cD A and then the 5' untranslated region
is incomplete lacking the initiation of translation.
The 3' end included the polyadenylation
signal and
the poly(A) tract, suggesting that the clone contains
the entire 3/-unstranslated
region of the mRNA.
The protein deduced from the cDNA coding sequence is 507 amino acids long without methionine.
This protein contains a highly hydrophobic peptide
region covering residues -18 to -1 (Kyte and Doolittle, 1982). The amino-terminal
amino acid, represented by serine, may correspond to the blocked
ond of the molecule, and is also confirmed by the
alignment
of known amylases.
The preprotein
should be processed to give a mature enzyme of 489
residues. From the open reading frame, a molecular
weight of 54,500 Da is predicted, which is comparable to that of other known amylases.
Tho level o[ polyrnorphism
was estimated by
using a polymera e chain reaction (PCR) amplification with two oligonu leotides, one in the sense
(OD 4), the other in the reverse sense (OD 7) of
the determined sequence cDNA.
Six bases were different in 10 different analysed
clones that corresponded
to two different amino
acids, alanine in tead of arginine in position 48
and tryptophan
insteacl of asparagine in position
87, confirming that at least two isoforms should be
pre ent.
Sequence

alignment

Phylogcnetic
relationship
were established with
humans, Drosophila, and shrimp as representative
of the animal group described by Raimbaud et al.
(1992) (Figure 4).lt appears that o-amylase of Pecten
is as near to human a to Crustacea or Drosophilo

230

S. Le Moine el al.

Table 1.

Summary

Crude exlracl
Acelone
pre pitate
In oluble tarch
Ultrafiltration

of the purificalion

procedure

of o-arnylaso

frorn Peclen maximus

digestive

gland.

Volurne
(mI)

Protein
(mg)

Activilv
)

pecific
activitv

1730
1460

8733
4724

814
771

0.09
0.16

1.0
1.8

100
95

10.80
0.29

203
40

18.80
138.60

206
1510

25
5

578
2

(I

1 IU of amylase activity is equivalont to 1 mg rnaltoso produced/3

o-amylases: the same percentages

caJculated (Table 2).

of similarity

were

Northern anaJysis
The purity and yi Id ofRNA were determined spectrophotometrically.
The ratio of absorbance at 260
and 280 nm was 1.97 for Trizol and 1.94 for isothiocyanate extracts. The two RNA extraction methods
gave similar quantitative
spectrophotometric
results for total RNA. Approximatel
3 mg total RNA
per gram wet weight was estimated. This value is
weak compared with that for rat (14 mg/g), but
higher than that for another mol! usk, Haliotis rufescens (1.7 mg/g) (Groppe and Morse, 1993a). Some
interfering substances may cause overestimaLion of
the spectrophotometric
value. Groppe and Morse
(1993b) identified abundant proteoglycans
in H.
rufescens that interfered with the performances of
oligo (dT) cellulose columns.
Autoradiograms
showed only one band at 1700
pb in total RNA. As ribosomaJ RNA 18S also migrates at this position, migration of messenger RNA
was performed to validate the sp cificity of hybridization. The same localization of bands (Figure 5)
confirmed a specific hybridization
of the amylase
cDNA probe to amylase RNA.

Discussion
Histologic and immunohistologic
studies (Henry et
al., 1993) showed that Pecten maximus amylase activity is located mainly in the stomach and digestive
gland. Recent investigations
of Giard et al. (1995)
demonstrated
that digestive cells of P. moximus secreted o-amylase in vitro at a low level. In vivo, it
accumulates in the crystalline style, where high specific activities of 2 to 3 units/mg of protein were reported in ChoromytiJus meridionoJis, for example
( eiderer and ewell, 1979). The specific activity of
the digestive gland crude extract (0.09 units/mg protein) is comparable to that reported by tark and

Purifi ation
factor

Yield
(%)

mino

Walker (1983) and Henry et al., (1993) for P.moxim us

(respectively, 0.14 and 0.33 units/mg protein). 01' by
Teo and Sabapathy (1990) for Perno viridis (0.25
units/mg protein). but it is low compared witb marine crustaceans: 0.5 units in Brachyourans and up
to 6 units in Penaeids. However, very low levels were
also reported in ephropsidae (Van Wormhoudt et
al.,1995).
lf we consider the specific a tivity of the purified
enz m , we can estimate an amyJase expression
level of 0.065% in the crude extract. This level is
higher than that found previously (Weber et al.,
1976; Henry et al., 1993), but the specific activity
of the pure amylase protein was weak in comparison
with commercially
purified amylase, and tbe percentage of amylase expression in the digestive gland
may be overestimated.
An inhibition of amylase
activiL by sugars still present after dialysis and
ultrafiltration
could cause this discrepancy.
Concerning purification, the method ofmodified
starch (Matsuura et al., 1978) gave in one step a
highly purified enzyme: a purification factor of 200
and a yield of 25%. However, after dialysis a high
level of sugar was measured, which probably resulted from partial hydrolysis
of the modified
starch, 01' poor elution of enzyme from its substrate.
Molecular data showed that amyla e of P. moximus
is expre sed at 0.02% in the digestive gland cDNA
library, confirming the low express ion compared
with amylase (0.2%) or chymotrypsin
expression
in the hepatopancreas
cD A library of the shrimp
Penoeus vonnomei (Van Wormhoudt and Sellos,
1996; Sellos and Van Wormhoudt, 1992) or chymotrysin-like express ion (11 %) in the intestine cD A
librar
of another molluskc,
Haliotis rufescens
(Groppe and Morse, 1993). Otber intestinal enzyme
xpre ion [sucrase-isornaltase
01' Jactase). however,
may have a low express ion (0.01% to 0.04%) in
adulL rabbit (Keller et al., 1992).
The abundance of sugars may have prevented deLermination of the amino-terminus
of the protein.

Amylase

in Pecten

maxirnus

231

the lower molecular weight of the molecule estimated from the nueleotide sequence which would be
around 55,000 Da, compared with that of60,OOO Da
measured by SDS electrophoresis. Most ofthe animal
amylases have this size: 53-55 kDa for human, rat,
pig, and insect. The calculated pI of 6.76 explained
the low migration in Da vis electrophoresis. In P.maximustwo isoforms have been detected, while in other
mollusks, fOUTisoforms were determined in Chloromytilus meridionalis (Newell et al., 1980) and in
Dreissena polymorpha (Scheil and Gunther, 1981)
and eight isoforms in MytiI us edulis (Sanger and Hagenmaier, 1986)-in this latest species only fOUTisoforms were expressed per animal. The mode of
expression of these isoforms and their significance
are not known in mollusks. In crustaceans and arnphipods the expression of the different isoforms is
controlled by carbohyclrate substrate (Borowsky,

Myosin

p-galactosidase
Phosphorylase

Bovine serum albumin

Figure 1. Electrophoresis
of o-amylase from P maxinlLlS
digestive gland in 7.5% polyacrylamide
gel. Lane a, proteins are stained with sil ver; lane b, amylase activity is
revealed on agarose overlay with iodine coloration. Electrophoresis was run at 40 mA/slab and 120 V for 180
minutes.

Ovalbumin

Carbonic anhydrase

However, a putative acetylation of the arnino-terminus serine, determined by translation of the cDNA
elone, and a putative glycosylation site (N-X-S/T)
present in the amino-terrninal
part of the protein,
may explain the inaccessibility
to Edman degradation. A post-translation
modification
may explain

Figure 2. Electrophoresis
of o-arnylase in 7.5% polyacrylarnide gel wilh 0.1 % sodiurn dodecyl sulfate (SDS).
Enzyme sample were treated with 4% SDS, 10% z-mercaploelhanol
for two minutes at 100C. Lane a, Pecten
maximus amylase; lane b, standard proteins; lane e, commercial pancreatic amylase.

egaa

ATG

CTT

TCA

CTG

ATT

ATA

GCC

GCC

TGT

TGT

GTA

ACC

GTT

GCA

AGT

AAT

CCG

ACC

TGC GCT CCT GGT

CAPGRNTIVHLF

CGC

AAC

ACG

ATC

GTG

CAT

AAA
K

TGG
W

ACA
l'

GAC

ATA
1

GCA
A

AAG
K

GAA
E

TGT
C

GAG
E

AGG
R

TTC
F

CTG
L

TTT TGT
FC

GGC
G

GTC

CAG

ATA

TCG
S

CCA
P

CCT
P

AAT
N

GAA
E

AAC
N

CGT
R

AGA CCA TGG

RPWWERY

TGG

GAA

AGG

TAT

CAG

CCG GTC
PVSYKL

AGC

TAC

AAA

GGC AGC GAG GAT CAA

GSEDQLRDMISRCNRVNV

CTA

AGG

GAC

ATG

TCC

AGA

TGT

AGA

ACC
l'

~
TLS
GAA
E
.

TGG
W
OON

GGC
G

GCT

GGG

55
-3

CTT

1'1'1'

109
16

GGA
G

CCG
P

AAT
N

163
33

CTT
L

GTC

AAT
N

AAC
N

217

CTG

CAA

ACA CGG
TRS

AGT

271
70

AAC

AGG

GTC

GTC

325

LAG

ATA
1

TAC

-+ODN

ACG
l'

CTT

GGG
G

ACA
l'

TCG
S
1

GAT

ATT

AAC

88
ATA
1

AAC
N

CAC
H

ATG
M

ACA
l'

GGA
G

GTC

GGT
G

GGA
G

TCA
S

GGG
G

379
106

GCG GGA TCA CAT

AGSHWNGDTLSYPGV

TGG

AAT

GGA

GAC

ACG

TTG

TCA

TAT

CCC

GGC

GTT

433
124

GTT

ODN 7 ~(

_
ATG
M

487

AGT
S

541
160

ACT

CAG

TAT

595
178

ATA GAC
IDA

GCC

GCA
A

AAA
K

648
196

ACA
r

CTG
L

CAT
H

GAT
o

CTA
L

/lAC
N

703
214

TTC

CAG

GAA

GTG

ATT

GAT

757

CCG
P

TTC
F

TCT
S

GCC
A

TGG
W

GAC

TTC
F

AAT
N

ACC
l'

GGA
G

AAT
N

GAG
E

TGT
C

CAC
H

AGC
S

TCC
S

GAT

AAC
N

ATC
T

CAT
H

GAT
D

TAT
Y

AAC
N

AAT
N

GCC
A

GAA
E

GAA
E

ATA
1

CGC
R

AAC
N

TGT
C

CGC
R

CTC
L

GTC

CTC
L

GCT
A

GAT

TTA
L

AAG
K

CTC
L

AGC
S

AAG
K

AAC
N

TAC
Y

GTC
V

CGA
R

GAG
E

GAA
E

ATT
T

ATG
M

AAC

CAT
H

CTG ATC
LID

GAC

TTG
L

GGC
G

GTG
V

GCT
A

GGA
G

TTC
F

CGA
R

CAT
H

ATG
M

TGG
W

CCT
P

GGA GAT
GOL

CTA

AGA
R

GCG
A

ATG
M

1'1'1'
F

GGC
G

TCT

GCT

GTA

1'1'1' GGT

ATC

ATG

GGA

GGT

GAA

GGA AGG AAA CCA

S
GRKPFIFQEVTD
-OON 2
CCA ATT AGT GCG TCG GAA

1'1'1'

AAC
N

TTC
F

AGT
S

TCG

TAC

ACA

GGT

ATC

GGC

CGA

GTC

TTC
F

CGG
R

AAT
N

GAA
E

AAC
N

AAG
K

GCC
A

GGC

ATG

CCA

AAC

TCG

AAT

GAC

GTC

919

286

GGA
G

CAT
H

GGA
G

GGA
G

GGA
G

GGT
G

GGT
G

973
304

AAA
K

CTC
L

GCC
A

ACA

GCC
A

TTC
F

ATG
M

TTG
L

1027
322

AGT
S

AGC

TAC

AAC

TTC

GAC

AGG

TCA
S

1081
340

ATC
1

1'1'1' GGT
F
G

GTT

AAA
K

CTA
L

GGA
G

CAA

GTC

AAC

CTT

CAC

AAC

TGG

GGC

GAG

GCT

TGG

ODN

6 .4------------------------

GTG GTG TTT

VVFIDNH

ATT

GAT

AAC

CAT

GAC
o

AAC
N

CAG
Q

CGA
R

CCT
P

TTG
L

ACC

CAC
H

TTC
F

GAA
E

CCA
P

CGC
R

CCC
P

TAC
Y

GCA
A

CAT
H

CCA
P

TAC
Y

GGC
G

1'1'1'
F

ACC

CGC
R

TTA
L

ATG
M

ACT

l'

AAC

GCT

GAC

CAG

GGT

GAC

CTC
L

ATC TAC AAT ATG

IYNMVAFRNIVMGQ

142

232

AAT

52

CCA

CCT

ACG

TGC

GTA

GCG

l'

S
Y
N
F
o
R
-------------------------+1

ACT

865
268

ODN 3

CAT

AAT

GGC

GAC

AAC

ATC

AAC

GAT

GTC

ACT

ATA

GGT

AAC

GGA

TGG

ACG

TGT

GAA

CAT

CGG

TGG

AGA

GAA

1189
376

TTC

AGG

AAC

ATA

GTA

ATG

GGA

CAG

AAT
N

CAA
O

CAC
H

1243
394

ODN

CTA
L

:,."(;..--

1135

358

GGT
G

AAC
N

TAC
Y

CAG

ATC
1

GCC
A

1'1'1' GGT
F
G

AGG
R

GGA
G

AAC
N

AAA
K

GG/I TTC
G
F

1297
412

ATG
M

/lAC ATG
N
M

GAC
o

AAC
N

CAC
H

AAC
N

CTC
L

GAC
o

CAA
Q

ACA
r

CTG
L

CAG

ACT
l'

GGC
G

CTT
L

1351
430

GCG
A

GGA
G

ACT
l'

TAT
Y

TGT
C

GAC

GTC
V

ATT
1

TCC
S

GGT
G

AGC
S

TAT
Y

GAT

GGT
G

TCT
S

AGT
S

TGT
C

l405

TCT
S

GGG
G

ACC
l'

GAG
E

ATC
T

CAG
Q

GTC
v

GGA
G

AAT
N

GAT
o

GGA
G

AAT
N

GCA
A

CAT
H

TTC
F

AGC
S

ATC
1

AGC
S

1459
466

AAC
N

AGT
S

AGC
S

GAT

GAC

CCT
P

ATG
M

ATC
1

GCT
A

ATT
1

CAC
H

GTT
V

GGC
G

GCG
A

AAA
K

AAA
K

GGT
G

CAG
O

1513
484

CCG
P

AAG
K

GTT
V

ACG
l'

ACG
l'

TGA

egttaeeeegattattageettaeeategaeatagggeeegtaaceac

TGG

TGG
W

GAC

w
ATC
1

GCT
A

CCC
P

AAT
N

811
250

l'

egtegeeatetttgttttateattgttetgeteaeaegaaattaaattg

tattagagataatatla),

448

1580
489
1645

Figure 3. Nucleotide
sequence of
Pecten o-arnylase cDNA and deduced
amino acid sequence. The amino acid
sequence is nurnbered sequentially
from the first amino acid of tbe rnature protein. The proposed
signal
peptide is underlined and in boldface
type. The nucleotide sequences corresponding to the synthetic oligonuc1eotides
(ODN)
used
in
the
sequencing
procedure
are underlined. The proposed polyadenylation
site is twice underlined. The putative
glycosylation site is shaded.

Amylase in Pecten maximus

PEC

SNPTCAP-GRNTIVHLFEWKWTD:AKECERFLGPNGFCGV

ISPPNENRLV--NN--RPWWERY

PVSYKL

PISYKLETRSG~EE

PEV

FDTNYASGRSGMVHLFEwKWDDlAAECENFLGPNGYAGV

VSPVNENAVK--DR--RPWW~RY

ORO

WDPNSS-N

VSPPNEYVEVY

IIUM

YSPNT

VIVHLFEWKWSOIAAECENFLGPRGf'AGV

LRDl'lSRCNRVNVRIYSDTVINHMTGVGGS-----

:0'>

FASMVKRCNAVGVRTYVDWFNHMAANGGTYGTGG

1I 1

OVKRPWWERY PVSYKLVTRSGDENA,KOI'(\l7RCNNVGVRIYVOAVINHMSGGWPM-----

GRTSIVHL,EWRWVDIALECERYLAPKG,GGVOVSPPNENVAI--YNPFRPWWERY

PI

TRSGSE

233

109

rVSYKLCTRSGNEDEFRN~TRCNNVGVRIYVDAVINHMCGNAVSA----

109

P2

PEC

GTG- TAGSHWNGDTLSYGVFPFSAWD,NTGNECH S SDMN1 HDYNNAE E l RNCRLVS LADLKLS KNYVRE El TOYMNH~ l DLGVAGFR l DAAJ(H~PGDLRAM FGT LHDLNSAV - FGS

220

PEV

-- - - ---STASPSSKSY

21 q

PGVPYSSLD,NPT-

-CAl R- - - - -NYNDANEVRNCELVGLRDLN
VRNCKLVGLNDLN

NSYVODKWE FLDH LI DLGVAG,RVDAAKHMWPADLAVI

DRO

GTGASGGSS FDSGAESY PGVPYSAFDFNDGN-CHTGSGN IENYGDAN

HUM

GTSSTCGSY FN PGSRDFPAV PYSGWDFNDGK-CKTGSGDI ENYN DATOVRDCRLTGLLDLALEKDYVRSK

loop3

PEC

GRKPFI ,OEVI DMGGEPI SASEYTGl GRVTN, l ,GVKLG

PEV

GSKAYIV

DRO

GARPFl F EVIDLGGEAISSGEYVGNGRVTEf"RYGKYLGEAFR--GNN

HUM

GSKPFlY

GTDYVRGKl REFMNKL l SYGVAGFRI DASKHMWPGOMKAI ,OSLDNLNTDF - f'KA

lAEYMNH LI DI GVAG,RLDASK HMWPGDr KAr LOKLHN LNSNW- FPA

PLTH ,EPRPYKLATArMLAH

LQYLTNWGTAWGFAASDRSLVFVDNHDNQRGHGAGGADVLTYKVPK
LKYLNNFGEGWGMIDRHDALVFIDNHDN

EVIDLGGEPIKSSDYFGNGRVTE,KYGAKLGTVIRKWNGEKMSYLKNWGEGWGFVPSDRALVFVDNHDN

PYGITRLMSSY

YKMASAFMLAHPFGTPRVMSS,

PEC

------NFDRSNT

-GPPHNGDNINDVTI-NADLTCGNGWTCEHRWREIYNMVA'RNIW.

PEV

-- - - -- - -SITDT

-GPPTTDGHN lAS Pl FNSDNSCSGGWVCEHRWRQIYNMVAFRNAVGSDEI

DRO

YW

HUM

RWPR F NGNDVNDWVGPPNNNGVIKEVTI

335
330

RGHGAGGDMILTFRVSKWYKMATAYMLAWPYGYTRVMSSY

338
340

P8

NLQHWWDNGNY lA,GRGNKGFIAMNMDNHNL

TLQTGLPAGTYCDVISGSY

NWWDNGSN ISFSRGSRGFVAFNNONYDLNSSLQTGLPAGTYCDVI

DKNDWIGPPHDGSFNIISPSFNADGSCGNGWrCEHRWRQIYNMVEFRNVAHGTDMNDWWDNGSN
-

RGHGAGGASILTNDARLYKMAVGFMLAHPYGF7RVMSSY

p7

P'>

WWEN

2~

224

loop3

V,R - -NENKASNLHNWGEAWGMPNSNDVWFlDNHDNORGHGGGGG-

EVIDMGGEAISKSEYTGLGAITEFRHSDSIGKVFR--GKN

YGRLKNLNTDHGFAS

SGSK

431
437

IAFCRGNKGFLAINNDGWDLKETLOTCLPAGTYCDVISGSK

456

PDTTCGNDWVCEHRWRQIRNMVI FRNWDGQPITNWYDNGSNQVAFGRGNRGFlVFNNDDWSFSLTLQTGLPAGTYCDVISGDK

4'>7

PEC

DGSSCSGTEI

VGNDGNAHFSISNSSDDPMIAIHVGAKKGQPKVTT

PEV

SGSSCTGKTVTVGSDGRASINIGSSEDDGVLAIHVNAKL

476

DRO

NGGSCTGKSVTVGGDGKAYI EITTMWDGVLAI

49'>

HUM

1NGNCTGIKIYVSDDGKAH,SISNSAEDPFIAIHAESKL

HANSKL

489

496

Figure 4. Amino acid sequence alignments of amylases of Pecten (PEe), shrimp (PEV, Van Wormhoudt and Sellos,
1996). human (HUM, Nakamura el al., 1984). and Drosophila (DRO, Boer and Hickey, 1986). Gaps have been inlroduced
in the s quences lo facilitale alignmenl . Po ition 1 relate to the matute amylase arnino-terminus,
Underlined sequences
correspond lo the secondary structure
lements comrnon to animal o-arnylases (Raimbaud el al., 1992).

Table 2. Percentage of sirnilarily of o-amylases frorn

different groups: Penaeus vannamei (EMBL = X77319);
Drosophila melanogaster (SW! SPROT = P08144): and
Horno sapiens (P04746).

Peclen
Peclen
Penaeus
Drosophila
Human

100

Penaeus

Drosophila

57

55

100

60
100

Hwnan
60
62
54
100

1984; Le Moullac et al., 1996). In insects, fOUT isozymes have been de cribed in relation to sex and development (Da Lage et al., 1992).
Only one P maximus cD As was characterized.
Its nucleotide sequence shows that the secreted protein contains 489 amino acids after processing of
an 18 amino acid hydrophobic signal peptide. The
thrce amino acids that belong to the active site of
the molecule are also present: Asp-193, Glu-229,
Asp-294. The consensus region that defines the ea-

234

S. Le Moine el al.

talytic clomain as (3/a) 8 barrels (Nakajima et al.,

1986; ]anececk, 1992, 1994), found in almost a11the
other amylases from clifferent sources, can also be
found in the P maximus amylase. These highly conserved regions are very likely either active or substrate sites. Ten cysteine residues werc dctermined
that could correspond to at least four disulfide bridges common to al! o-amylases. Compared with vertebrates, we noticed the absence of three residues
of cysteine in positions 71, 104, ancl 115. As one
more bridge is observed in the porcine amylase (Pasero et al., 1986), linking cysteines in positions 71
and 115, one could exist in Pecten ttiaximus, between residues 5 and 37.
The same percentage identity found with vertebrates and arthropoda may indicate a convergence
(Baba et al., 1981) or more likely a slow evolution
from a common ancestor. More sequence determinations arnong the animal kingdom are necessary to
validate this hypothesis, to show evolutionary relationships at different levels with biologica11y active
molecules, rather than with structural molecules.
For that purpose, o-arnylase may be a good model.
In common with Drosophilo and human o-arnylase,
we specially noticed a gap of five arnino acids in
position 101. In common with crustacean o-amylase, the absence of six al' eight arnino acids in position 330 is noticeable. The carboxyl-terrninus
of
Pecten presented 8 extra amino acids, but the signif-

1700

bp

icance of these changes in terms of function

yet unclerstood.

Experimental

10 20

mRNA

total

30 J.lg
RNA

Figure 5. Northern blot analysis of AMY sequences in

total RNA and mRNA extracted b Trizol frorn P. maximus
digestive gland. Samples were denaturated
by DMSOI
glyoxal treatment
before agarose electrophoresis
and
transfer onto nylon membranes. Left, 1, 2, and 5 J-gof
mRNA poi)' (A): right, 10, 20, 30 J-gof total RNA.

Procedures

Animals
Animals were clredged in the Bay of Brest and either
directly used (cDNA library) al' maintained during
one week in experimental tank with a regular food
supply (amylase purification and RNA expression)
to ensure good expression of digestive enzymes.
Their cligestive glands were dissected and quickly
frozen in liquid nitrogen. The frozen digestive
glancls were crushed to afine powder with a Dangoumeau rnill. For cDNA library and RNA analysis,
a11material for dissection and homogenization
was
sterilizecl and a11 solutes were treated with diethyl
pyrocarbonate
(DEPC) to avoid RNase contamination before and during RNA extraction.
Assay of a-amylase
Amylase activity was assayed by determination
of
starch hydrolysis according to iodine reaction: 0.2
mi of enzymc extract was added to 0.5 rnl of 0.1 %
soluble starch in 0.2 M acetate buffer, pH 6.2, containing 0.2 M NaCl and 0.02 M CaCl2, and the mixture
was incubated at 45C. This pH was optimal for Pecten maximus (Stark and Walker, 1983). After 10 minutes of incubation,
the remaining
starch was
deterrnined by iodine reaction. One unit of o-amylase activity was defined as the amount of enzyme
that degrades 1 mg 01' starch per minute at 45C (Samain et al., 1977). It can be estimated that 1.53 units
of arnylase expressed in milligrarns 01' hydrolyzed
starch per minute are equivalent to 1 unit expressed
in milligrams of maltose per three minutes.
The proteins were determined according either
to the method of Lowry et al. (1951) al' to the micromethod ofBradford (1976) for very low protein concentrations
(Bio-Rad, Ivry/S ine, Fran el. Bovine
serum albumin was used as a standard.
Purification

125

is not

of a-amylase

Any step of the purification

procedure was perforrned at 4C. The digestive glands were hornogenized in 10 mM calcium acetate buffer containing
20 mM sodium chloride, pH 6.5. The homogenate
was centrifuged at 10,000 g for 20 minutes, ancl the
pellet was discarded. The particule-free supernatant was filtered through glass-wool to remove the
overlaying lipids. This suspension was ultracentrifuged at 30,000 g for 45 minutes, and the pellet was

Amylase

also discarded. The supernatant (crude extract) was

treated with 2 volumes of acetone for 30 minules.
After centrifugation,
the pellel was resuspended
in
a similar volume of e lraction buffer.
Affinity chromatograph
was carried out on
tarch in olubilized
by ammonium
sulfale, according to the method of Minamiura el al. (1975)
and Matsuura et al. (1978). Starch was heated al
70C for 30 minutes in 10 volumes (w/v) of a 15%
ammonium
sulfate solution,
pH 7.8, and then
cooled. The enzyme extract was added lo this solution and allowed to stand overnight at 4C with
gen tle stirring in order to adsorb the amylase. The
amylase-starch compl x was collected by centrifugation at 4C. Amylase was recovered by incubation
at 40C for one hour in a solution adjusted al pH
6.5, which contained 20 mM sodium chloride and
10 mM calcium acetate. The supernatant
solution
conlaining the liberated amylase was obtained after
centrifugation
at 10.000 g. Thi la 1 step wa repeated five times. The supernatants were mixed and
dialyzed against disti11ed water for three hours,
three times. The extract was then lyophylized. An
ultrafiltration
by centrifugation
(MC, Millipore,
Saint Quentin, Yvelines, France) allowed partial
elimination of sugars a sociated with extracts.
Electrophoresis
The purity of amylase was assessed by electrophoresis 011 7.5% polyacrylamide
gel in Tris buffer, pH
8.3, both in native (Oavis, 1964) and denaturing
SOS (Weber and Osborne, 1969) conditions. After
migration the slabs were cut into two sheets. One
sheet was placed in clo e conlact with 1% agarose
gel containing 1% soluble starch to detect arnylase
activity after iodine coloration. The other sheet was
treated with sil ver, lo stain proleins.
Myosin (205 kDa), 3-galactosidase
(116 kDa),
phosphorylase b (97 kDa), albumin bovine (66 kDa),
ovalbumin (45 kDa), and carbonic anhydrase (29
kDa) were used as molecular weight standards.
R A isolation
Total RNAs from digestive gland were extracted according to two protocols. The fir t one a110w d a
rapid extraction of total RNA from 50 to 100 mg
lissues b Trizol (Life Technologi
, Cergy Pontoise,
France), the second one was the cla ic method
u ing guanidinium
isothiocyanale
(Chirgwin et al.,
1979). Oigestive glands were disrupted in liquid
nitrogen with a grinder. The powdered tissue was
immediately dissolved in a 4 M guanidine thiocyanate (5 ml/g oftissue) solution. RNA concentrations

in Pecten

maximus

235

were measured al 260 nm using the conversion faclor 100 = 40 ..g/mlRNA. R A messenger poly(A)+
wa
eparated from total RNA by chromatography
on oligo (dT)-cellulose (Stratagene, La Jolla, Calif.,
U.S.A.).
1

oithem and dol blot hybridization

RNAs were fractionated either by agarose-formaldeh de gel electrophoresis

or after glyoxal/OMSO
treatment
and transferred
pa sively onto nylon
membrane (Hybond
-, Amersham, Buckinghamshire, U.K.). For dot blot, samples of total (10 lo 30
ug) and mes enger (1 to 5 ..g)RNAs were dissolved
in J 1 ..Iof water and denaluraled by adding 5 u.l of
10 x MOPS, 9 ..lof 12.3 M formaldehyde, and 25
..lof Jormamide, incubated 15 minutes at 55C, and
dlutod in 10 X SSc. They were deposited onto
n Ion membrane, using the Schleicher & Schuell
(Ecquevilly,
France) minifolel apparatus.
Membranes were rinced twice with 10 X SSC and dried
a180 fortwohour
to fixRNAbeforehybridization.
Pecten maximus cO A amylase was labeled with
12p b
random priming using a-dATP (2000 Ci/
mmo1) and the Klenow O A polymerase (Hexaprimer kit, Appligene, Illkirch, France). The prehybridization-hybridization
conditions applied were
50% formamide,
aCl 1 M, salmon ONA 0.5 mg/
mI, 1% SOS, at 42C. Washings ofmembranes
were
2 X SSC, 20C, five minutes (twice). 2 X SSC, 0.1 %
SOS, 60C, 30 minute (twice), 0.1 X SSC, 20C, 30
minutes (twice).
orthern membranes were autoradiographied
and then sliced around the band of
interest for scintillalion
counting. Oot membranes
were cut with the cutling template (Schleicher &
Schuell) lo obtain individual squares of the filtered
area, ancl each portion of membranes was introduced in a scintillaton vial with Filter Count (Packarel Instrurnonts, Rungis, France) scintillation liquid
to evaluale quantitative hybridizalion
levels.
Complementary
procedures

D A library and screening

The mRNA from one digestivo gland of Pecten maximus was e tracted by the RNA i olation kit of Stralagene. A A Z P cO A library was established
following the manufaclurer's
protocol (Stratagene).
This protocol produced
cO As that were then
cloned in the polylinker of pBluescript between
EcoRI and Xhol. Five million independent
phage
were obtained, and after amplification
the library
titration was 101Opfu/ml. A crustacean cONA amyla e probe (Van Wormhoudt and Sello, 1996) was
used lo screen P maximus digestive gland cONA

236

S. Le Moine el al.

library. This probe was labeled with 32 P by randorn

priming using a-dATP and tbe Klenow D A polymera e (Hexaprimer
kit, Appligene).
The phage
cD As were transferred
onto nylon membranes
(Hybond
" Amersham), and the membranes were
treated, as previously reported, for tbe
ortbern
blots. A prehybridization
wa performed in 40%
formamide, 1% SDS, aCI 1M, salmo n D A 0.05
;..g/ml finaJ concentration,
for 4 hours at 40C. After
denaturation
at 100C, five minutes, the labeled
probe (106cpm/ml) was added to a new olution,
and hybridization
took place at 40C for 16 hours
under agitation. After washing, autoradiograms
revealed positive clones. A protocol of in vivo excision using a helper phage gave bacterial colonies
that contained
tbe double-strand
pBluescript
phagemid
with the clon d cDNA inserts ( tratagene).

Acknowledgments
This study was supported by financial grants from
the Region Bretagne. The authors are indebted to
C. Langdon (Oregon State University) for improving
the English.

References
Baba, M.L., Darga, L.L.. Goodman, M., and Czelu niah, J.
(1981). Evolution of the c tochrome c investigaling the
maximum parsimony rnethod. , Mal EvoI17:197-213.
Bayne, B.L. (1976). Marine mussels, Their Ecology and Physiology. London, U.K.: Cambridge University Press.
Boer, P.M., and Hickey, D.A. (1986). Tbe o-amylase gene in
Drosophila melonogaster,
nuc1eotide sequence, gene
structure and expression. Nucleic Acid Res 14:8399-8411.
Borowsky, R. (1984). Environmenlal control of amylase phenotype in arnphipods of the genus Gammarus Biol BuJl
167:647-657.
Boucaud-Camou, E.. Lebe nerais, c.. Lub t, P., and Lihrmann,
1. (1983). Dynamique et enzymologie de la dige tion chez
l'huitre Crossostrea gigas (Thunberg). In: Ifremer, Actes
de colloques. Montpellier, France: Bases biologiques de
l'aquaculture, 1:75-96.
Bradford, M. (1976). A rapid and sensitive method for the
quantitalion of microgram quantities of protein utilizing
the principie of protein-dye binding. Anal Biochem
72:248-254.
Chirgwin, J.J., Przbyla, A.E., Mac Donald, R.J., and Rutter,
w.J. (1979). Isolation ofbiologicaly active ribonuc1eic acid
from sources enriched in ribonuc1eases Biochemistry
18:5294-5299.
Dagorn, J.C., and Mongeau, R. (1977). Different action of hormonal timulation on the biosynthe is of three pancreatic
enzymes. Biochim Biophys Acta 498:76-82.
Dagorn, J.c.. and Lahaie, R.G. (1981). Dietary regulation of

pancreatic prot in synthesis, 1: rapid specific modulation

of enzyme synthesi by hanges in dietary composition.
Biochim Biophys Acta 654:111-118.
Da Lage. .L., Lemeunier. F., Cariou, M.L., and David, J.R.
(1992). Multiple analysis genes in Drosophila anana sae
and related species. Genet Res 59:85-92.
Davi ,B.T. (1964). Disc electrophorcsis, 11:method and application to buman serum proteins. Ann
Y Acad Sci
321 :404-428.
Desnu IIc, P., Reboud. .P., and Ben Abdeljlil. A. (1962). Influence of the compo ition of the diel on the enzyrne conlent
of rat pancreas. In: De Reuck A.V.S, and Cameron, M.P.
(eds). Giba Foundalion Symposium on the Exocrine Pancreas. Boston, Mass.: Little, Brown, 90-114.
Dessen, P., Fondrat, c., Valencien, c., and Mugnier, C. (1990).
BISANCE, French service for access to biornolecular sequence databases. Gomput Appl Biosci 6:355-356.
Giard, W., Favrel, P., and Boucaud-Carnou, E. (1995). In vitro
investigation of o-arnylase release from Ihe digestive cells
of!he bivalve mollusc Pecten maximus, effect of second
messengers and biogenic amines. , Gomp Physiol B
164:518-523.
Groppe, .C.. and Morse, D.E. (1993a). Mollu can chymotrypsin-like prolea e, tructure, localization and substrate
sp ificity. Arch Biochem Biophys 305:159-169.
Groppe, J.c., and Mor e, D.E. (1993b).lsolation ofa full-length
RNA ternplates for reverse tran cription from tissues rich
in
Ase and proteoglycans. Anal Biochem 210:337-343.
Grossman, G.L., and [ames. A.A. (1993). The salivary gland
of the vector mosquito Aedes aegypti express a novel rnemb r of the amylase gene family. Insect Mol BioI1:223-232.
Hein, [. (1990). Unified approach to alignment and phylogenies. Methods EnzymoI183:624-644.
Henry, M., Benlimane,
., Boucaud-Camou, E., Ma!hieu, M.,
Donval. A., and Van Wormhoudt, A. (1993). The amylase
secreting cells of the stomach of the scallop, Pecten
maximus, ultrastructural. immunohistochemical
and immuno ytochemical
characterizations.
Tissue
Gell
25:537-548.
Ianecek, S. (1992). New conserved aminoacid region of aamylase in the third loop of their (3/a) 8 barre! domain.
Biochem '288:1069-1075.
[anecok. S. (1994). Sequence similarities and evolutionary
relationships of microbobial. plant and animal o-arnylases. Eur ] Biochem 224:519-524.
Kell r, P., Zwicker, E., Mantei,
., and Semenza, G. (1992).
The !evels of la ta e and sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level
and post-translationally. FEBS Lett 313:265-269.
Kyte, J., and Doolittle. R.F. (1982). A single metbod for displaying the h dropatbic cbaracter of a protein. , Mol Biol
157:105-132.
Korc, M., Owerbach, D., Quinto, C; and Rutter, W.J. (1981).
Pancreatic islet-acinar cell interaction, arnylase messenger RNA levels are determined by insulin. Science
213:351-353.
Le Moullac, G.. Van Wormhoudt, A., and Aquacop. (1994).
Adaplation of digestive enzymes lo dietary protein. carbohydrate and fibre levels and influence of protein and
carbohydrate quality in Pennaeus vannamei larvae (Crustacea. D capoda). Aquat Living Resourc 7:203-210.
Lowry, O.H .. Rosebrough,
.J., Farr, A.L., and Randall, R.J.

AmyJase in Pecten maximus

(1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.
Maniatis, J., Fritsch, E.F., and Sambrook, J. (1989). Moleeular
Cloning: A Loborotory Manual. Cold Spring Harbor, .Y.:
Cold Spring Harbor Laboratory.
Mathers, .F. (1973). Carbohydrate digestion in Ostrea edulis
L. Prae Malee Soe 40:359-367.
Matsuura, K., Ogawa, M., Kosaki, G., Minamiura,
., and
Yamamoto, T. (1978). o-Amylase from human pancreatic
juice as an electropboretically
pure i ozyme. J 8ioehem
83:329-332.
Minarniura,
., Kimura, Y.. Tsujino, K., and Yamamolo, T.
(1975). Isozymes of o-amyla e in human urine. J 8ioehem
77:163-169.
akajima, R., Imanaka, J., and Aiba, S. (1986). Nucleotide
sequence of the 8aeillus stearohermophilus
o-arnylase
gene. Appl Microbiol Bioteehnol 23:355-360.
Nakamura, Y.. Ogama, M., Nishida, T., Emi, M., Kosaki, G.,
Himeno, S., and Matsubara, K. (1984). Sequence of cDNAs
for human salivary and pancreatic o-amylases. Gene
28:263-270.
Newell, Re., Parker, 1., and ook, P.A. (1980). A possible
role of o-amylase isoenzymes from the style of the mussel
Chloromytilus meridionalis (Krauss) following thermal acclimation. J Exp Mar Biol EeoI47:1-8.
Pasero, L., Mazzei-Pierron, Y., Abadie, B. Chicheportiche,
Y., and Marchis-Mouren, G. (1986). Complete amino-acid
sequence and location of the five disulfide bridges in porcine pancreatic o-amylases. Biochim Biophys Acta 869:
147-157.
Paynter, K.T., and Chen, T.T. (1991). Biological activity of
biosynthetic rainbow trout growth hormone in the eastern
oyster, Grassostrea gigas. Biol 8ulJ 181:459--462.
Pictet, R, Mc Donald, R.J., Swain, W.F., Grebar, M.M., Hobart,
P.M., Crawform, R, Shen, L.D., Bell, G., and Rutter, W.J.
(1981). Differenliation of the pancreas, an analysis of the
structure of the arnylase and insulin genes. Fortsh Zool 8
26:227-245.
Raimbaud, E., Buleon, A., Perez, S., and Henrissat, B. (1992).
Hydrophobic cluster analysis of the primary sequences of
o-amylases. Int J Biol Macromolll :217-225.
Rutter, WJ. (1980). Structure of a family of rat amylaso genos.
Nature 287:117-122.

237

amain, J.F., Daniel, J.Y., Le Coz, J.R. (1977). Trypsine, arnylase et protenes du zooplancton, dosage automatique et
manuel. J Exp Mar Biol EcoJ 29:279--289.
Samain, J.F., Daniel. J.Y., Le Coz, J.R., Michaud, F., and Moal,
J. (1991). Digestive enzymes during the development of
Peclen maximus. Presented al the Eighth Internatinal Pectinid Workshop, Cherbourg, France.
Sanger, R., and Hagenmaier, H.E. (1986). Amylases in Mytilus
edulis (Mollusca Pteromorpha), ein nachweis zweir polymorph system. Zool Anz 217:116-118.
Scheil, H.G., and Guenther, A. (1981). Amylases in Dreissena
polymotpha
Pall (Mollusca, Eulamellibranchiata),
eviden e for two polymorphic systems. Zool Anz 207:3--4.
Seiderer, L.J., and Newell, R.C. (1979). Adjustrnent of the
activity of o-amylase extracted from the style of the black
mussel Choramytilus metidionatis (Krauss) in re ponse to
therrnal acclirnatation. J Exp Mar Biol EcoJ 39:79-86.
Sellos, D., and Van Wormhoudt, A. (1992). Molecular cloning
ofa c-DNA that encodes a serine protease with chyrnotryptic and collagenolytic activities in the hepatopancreas of
the shrirnp Penaeus vannamei (Crustacea, Decapoda).
FEB Lett, 309:219-224.
Stark, J.R., and Walker, RS. (1983). Carbohydrate digestion
in Peeten moximus. Comp Biochem PhysioJ 76B:173-177.
Teo, L.H., and Sabapathy, V. (1990). Preliminary report on
the digestive enzymes present in the digestive gland of
Perna viridis. Mar Bio/106:403--407.
Van Wormhoudt, A., and Sellos, D. (1995). Molecular cloning
and sequencing of three cD As that encode arnylase in
the hepatopancreas of the shrimp Penaeus vannaemei. f
Mol Evol 42:543-551.
Van Wormhoudt, A., Bourreau, G., and Le Moullac, G. (1995).
Amylase polymorphism in crustacea decapoda, electrophoretic and irnmunological studies. Biochem Systetnatics Ecol 23:139--149.
Weber, K., and Osborne, M. (1969). The relationship of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. f Biol Chem 244:4406--4412.
Weber, M., Fogli tti, M-J., and Percheron, F. (1976). Purification d'o-amylases par chromatographie d'affnte sur amidon feticue. Biochimie 58:1299-1302.