Characterization of a cathepsin L-associated protein in
Artemia
and its relationship to the FAS-I family of cell adhesion proteins
Alden H. Warner
1
, Ervin Pullumbi
1
, Reinout Amons
2
and Liqian Liu
1
1
Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada;
2
Department of Molecular Cell Biology,
Sylvius Laboratory, Leiden, the Netherlands
We reported previously that the major cysteine protease in
embryos and larvae of the brine shrimp, Artemia franciscana,
is a heterodimeric protein consisting of a catalytic subunit
(28.5 kDa) with a high degree of homology with cathep-
sin L, and a noncatalytic subunit (31.5 kDa) of unknown
function. In the study reported here the noncatalytic subunit,
or cathepsin L-associated protein (CLAP), was separated
from cathepsin L by chromatography on Mono S and
found to contain multiple isoforms with pIs ranging from 5.9
to 6.1. Heterodimeric and monomeric cathepsin L showed
similar activity between pH 5 and 6.5, while the heterodimer
was about twice as active a s m onomeric cathepsin L below
pH 5. The heterodimer w as more stable than the m onomer
between pH 6 a nd 7.4 and at 30–50 °C. Artemia CLAP and
cathepsin L are p resent in nearly equimolar amounts at all

[10], i mplantation [ 11], a nd molting [3,12,13]. In developing
embryos, cysteine proteases are often found in the cyto-
plasm a nd extracellular matrix where they may have
regulatory functions, unlike in somatic cells of multicellular
organisms where these enzymes are primarily lysosomal
and thought to play a role i n intracellular protein turno ver
and degradation [14,15]. In mammals, cysteine proteases
may function in transcription factor regulation [16], in
antigen processing [17], and in several parasitic organisms
cysteine proteases are considered to be virulence factors
because they are secreted at the site of invasion [18,19].
Over-expression and sec retion of c ysteine proteases is also
common in various pathological conditions [20–22].
In embryos and larvae of the brine shrimp, A. franciscana,
the major protease is a heterodimeric cathepsin L-like
protease (CLP) consisting of a catalytic subunit (CL) of
28.5 kDa and noncatalytic subunit of 3 1.5 kDa with a total
molecular mass of 60 kDa [23,24]. The catalytic subunit of
the complex has a high degree of homology with cathepsin L
from several sources [24]. T he noncatalytic subunit (cathep-
sin L-associated protein; CLAP) has, in vitro, a high affinity
for monomeric CL, and together, they form a heterodimeric
protease which has been resolved into seven isoforms with pI
values ranging from 4.6 to 6.2 [24]. Both subunits of CLP are
glycosylated; t he catalytic subunit contains O -linked carbo-
hydrates and the noncatalytic subunit contains N-linked
carbohydrate [24]. Cell fractionation and immunocyto-
chemical studies of Artemia embryos and larvae indicate
that about 85% of the protease is nonlysosomal with
considerable antibody stain a ppearing at the surface of y olk

embryos i s unique and functions outside lysosomes, in the
cytoplasm and extrace llular matrix, unlike CL in many
other higher eukaryotes.
Materials and methods
Purification of cathepsin L-like protease
The c athepsin L-like protease (CLP) in embryos o f the
brine shrimp, A. franciscana was purified using a m odifica-
tion of a published method [24]. Fifty grams of fully
hydrated Artemia cysts were homogenized in ice-cold
homogenization buffer (50 m
M
Tris/HCl, pH 7.2, 5 m
M
KCl, 1 m
M
dithiothreitol and 10 m
M
MgCl
2
)usinga
motorized mortar and pestle (Torsion Balance Co, Clinton,
NJ, U SA). Following centrifugation to remove nuclei, yolk
platelets, mitochondria (10 000 g, 20 min) and ribosomes
(105 000 g, 2.5 h), the soluble material was treated with
solid ammonium sulfate t o obtain t he 35–75% a mmonium
sulfate insoluble material. The latter was collected by
centrifugation, d issolved in Buffer A [15 m
M
potassium
phosphate, pH 6.8, 25 m

M
dithiothreitol, and 35–100 pmol of enzyme. The reaction
also contained dimethylsulfate (1.0–1.5%) in which the
substrate was dissolved. At the desired incubation time an
aliquot of the reaction mixture was added to an equal volume
of coupling buffer [5 m
M
mersalyl a cid, 30 m
M
NaOH, 2%
(v/v) Brij a nd 0.81 m
M
EDTA, adjusted to p H 4.0 with 1
M
HCl] to which w as added a n a dditional volume of coupling
buffer containing 0.5 mgÆmL
)1
Fast Garnet (Sigma, Mis-
sissauga, ON, Canada). After 15 min incubation at room
temperature, the complex wasextracted with 1 mL n-butanol
and the color intensity determined by analysis at 520 nm.
The number of pmoles of cathepsin L were determined b y
titration of t he acti ve site with E-64 as described previously
[29]. The concentration of heterodimeric cathepsin L was
64–65% of that calculated from the protein concentration,
while monomeric cathepsin L was 60–61% of the calculated
value based on protein content. Rate constants were
calculated as pmol b-naphthylamine released per minute
per pmol of active protease at p H 5.0 and temperature indi-
cated. Artemia p26 protein was a gift of T. MacRae

was treated with phosphatidylinositol-specific phospho-
lipase C (PI-PLC) (Sigma) prior to analysis by IEF to test
for glycosyl-phosphatidylinositol (GPI) units in the protein
[31].
SDS/PAGE was performed in 12% (w/v) acrylamide gels
[32]. Following electrophoresis, gels were stained for 1 h
with 0.1% (v/v) Coomassie blue R-250 in 40% (v/v)
methanol and 10% (v/v) acetic acid then destained
overnight in 5% (v/v) me thanol and 7.5% (v/v) acetic acid.
Acrylamide gels containing various preparations of CLP
and its subunits were also stained with Pro-Q Diamond
phosphoprotein stain (Molecular Probes, Eugene, OR,
USA) according to the manufacturer’s instructions.
Ó FEBS 2004 Cathepsin L and cell adhesion protein in Artemia (Eur. J. Biochem. 271) 4015
Cysteine protease analysis at different stages
in the
Artemia
life cycle
Harvested organisms were reared in the laboratory to the
desired stage in their life cycle [3,33]. At the desired stage,
intact organisms were washed with distilled water, blotted of
excess water then frozen by immersion in liquid nitrogen.
Ovisacs from adult females containing encysted embryos or
nonencysted embryos were removed with a scalpel while
frozen in liquid N
2
. Gravid f emales from which the ovisacs
had been removed were saved for analysis. Immature,
nongravid females containing no visible signs of eggs, and
adult males, w ere collected, washed a nd frozen in liquid N

Investigaciones Biome
´
dicas, CSIC/UAM, Madrid, Spain).
The library was constructed in phage kZAP II ( Stratagene,
La Jolla, C A, USA) with the cDNAs w ere inserted between
the EcoRI and XhoI sites in the multiple cloning region of
the vector. The phage were amplified in XL1-Blue-MRF¢
(Stratagene) then probed with a
32
P-labeled 564 bp PCR
product generated using primers constructed from amino
acid sequence data of CLAP, and c loned i nto p CR2.1
(Invitrogen, Burlington, ON, Canada). Approximately
2 · 10
6
plaques w ere screened using standard protoco ls
[34], and six plaques, identi fied by h ybridization to the
564 bp P CR product, were chosen for f urther analysis.
The isolated phage were converted to Bluescript phage-
mids using ExAssist h elper phage and a protocol provided
by the supplier (Stratagene). Six cDNA clones were grown
overnight in the presence of ampicillin (50 lgÆmL
)1
)andthe
DNA was isolated using the Wizard M iniprep Kit (Promega,
Madison, WI, U SA). All c lones showed identical restriction
maps, and two were sequenced by cycle sequencing using
primers c onstructed f rom the original 564 bp PCR product
and from information in the Bluescript phagemid. Sequen-
cing was performed on a Visible Genetics (Suwanee, GA,

The protein composition of Mono S fractions a–f were
analyzed by SDS/PAGE (Fig. 2). The main protein in
fraction a was the catalytic subunit o f 28 kDa, while peaks
or areas labelled b, c and d contained both subunits.
Column fractions b, c and d probably represent specific
undissociated isoforms of the heterodimeric CLP, as each
contained both subunits of the native protease. Gel lanes e
and f contained mainly CLAP of molecular mass 31.5 kDa.
The residual protease activity in peaks e and f disappeared
during re-chromatography on Mono S (Fig. 2B). Treat-
ment of an SDS/PAGE gel containing CLP and CLAP with
a phosphoprotein stain did not reveal phosphate additions
to these proteins. Only lanes in the gel containing the known
phosphoproteins ovalbumin, b-casein and pepsin gave a
reaction. Thus, while Artemia CLAP fractions e and f are
clearly distinguishable on Mono S, they have identical
molecular masses (31.5 kDa), and they are devoid of
phosphate linked to Ser, Thr and Tyr.
Analysis of
Artemia
CLAP by isoelectric focussing
CLAP fractions e and f (Fig. 1B,C) were analyzed by I EF.
Fractions e and f showed three and four bands, respectively,
on IEF gels with p I v alues r an ging from 5.9 to 6 .1 (Fig. 3).
Fractions e and f have at leas t one unique isoform e ach ( pI
5.9 for e and pI 6 .1 for f), while two bands of pI 5.95 and pI
6.0werecommontoeachofthemajorCLAPfractions,
although this does not mean that these are identical
isoforms. Overall, Artemia CLAP appears to contain four
isoforms in nearly equal a mounts, but these isoforms were

Mono S (Fig. 1B,C). The (0) at the bottom shows the absence of
protease ac tivity in e and f after re-chromatography. M w, molecular
mass ma rker.
Fig. 3. Isoelectric focusing of CLAP. Twenty-fi ve micro grams of
CLAP fractions e and f (Fig. 1 B,C) in a volume of 100 lLwere
applied to the top of separate glass tubes containing 6% acrylamide as
described in M aterials and m ethod s. Tubes containing pI standards
and column buffer only were prepared. After the proteins reached their
equilibrium positions, the gels containing the CLAP e, f, pI standards,
and buffer only were removed from their glass tubes, soaked in distilled
water for 5–10 min then stained with silver reagent. The pI values
assigned to bands in columns e and f were determined from b oth IEF
standards (Std) and buffer control gelruninparallel.Thenumbersat
the right represent the pI values of the major bands in e and f, while the
numbers at the left are the pI values of standard proteins. The arrow at
the right represents the pI value of 6.84 calculated for the unmodified
CLAP p rotein based on its d educed amino acid composition.
Ó FEBS 2004 Cathepsin L and cell adhesion protein in Artemia (Eur. J. Biochem. 271) 4017
2-fold higher activity at pH 4.3–4.7. Preincubation (1 h at
30 °C) of Artemia CL at pH 6.0 and 7.4 resulted in 85%
and 95% loss of cathepsin L activity, respectively, com-
pared to CLP which was less affected by these treatments
(Fig. 4B). Also, the monomer was completely inactivated
after 2 h preincubation at 40 °C and pH 6.8, whereas the
dimer retained about 70% of its initial activity under these
conditions (Fig. 4C). Similar differences in cathepsin L
activity were observed at a ll incubation temperatures
between 40 and 53 °C (data not shown). Overall, the CLP
complex is more stable than CL below pH 5, and between
pH 6.0 and 7.4 at temperatures exceeding that found in

cathepsin L at different pH and temperatures. (A) CLP (dimer) and CL
(monomer) were assayed at d ifferent pH f or cathepsin L activity (rate
constants). Each reaction vessel contained 40–60 pmoles of the a ctive
protease. (B) Different forms of the protease (solid bars, CLP; unfilled
bars, CL) were incubated for 1 h at 30 °Cin25m
M
KCl, 10 m
M
sodium phosphate, 10% glycerol and 0.2 mgÆmL
)1
BSA at the pH
indicated, then assayed f or cathepsin L activity at pH 5 .0 a nd 30 °C
and the rate constants determined. The control represents CL
(monomer) a nd CLP ( dimer) maintained at 0 °C and pH 6.8 prior t o
the assay. (C) Incubation vessels were set up to contain 80–100 pmoles
of CL (m on omer) a nd CL P ( d imer) in pH 6.8 buffer as described in
(B). The vessels were incubated at 40 °C and aliquots were removed at
30 min intervals, assayed f or cathepsin L activity at pH 5.0, and their
rate constants d etermined.
Fig. 5. Sensitivity of various proteins to Artemia cathepsin L monomer.
Reaction vessels contained 50 m
M
sodium acetate, pH 5.0, 0.5 m
M
dithiothreitol, 2.4 lg of CL (monomer), and 12–14 lg of CLAP, BSA,
artemin or p26 in a final v olume of 4 0 lL. A fter 0 and 60 m in incu-
bation at 30 °C, 10 lL were t aken from each reaction ve ssel for ana-
lysis by SDS/PAGE on a 12% gel. The numbers above each lane
represent the incubation time of the monomer with proteins shown
above each lane. Left lane (mw) contains molecular mass standard

differed from clone 1 mainly in that it lacked a 68 nucleotide
sequence at the 5¢ end, including sequence coding for the
first 15 amino acids in clone 1. Also, at position 568 in clone
2 an A was substituted for a G changing the amino acid
from R t o K. B oth cDNA clones have a short 5¢
untranslated region, and extensive 3¢ untranslated regions
rich in A + T, representing nearly 45% of the mature
transcripts. The 3¢ UTR of c lones 1 and 2 are composed of
about 72% A + T and differed f rom each other by 2.1%.
Also, c lone 1 c ontains seven consensus AT-rich m otifs,
while clone 2 contains five AT-rich motifs. Both clones
contain several putative pol y(A) addition signals ( AAT
AAA and ATTAA). The nucleotide sequences of clone 1
and 2 have been entered into t he NCBI database with
accession numbers A Y307377 and A Y462276, r espectively.
Starting from the amino terminus of the mature protein
(E44) (Fig. 7), a calculated molecular mass of 32.3 kDa and
pI of 8.0 were obtained using
EXPASY
(http://www.
expasy.org/) if the m ature protein te rminated at Q332.
While the calculated molecular mass is close to that
observed by SDS/PAGE (31.5 kDa), the pI value is
distinctly different from the values (5.9–6.1) obtained by
IEF for mature CLAP. These observations suggested
further post-translational modifications occur, leading to
mature CLAP. A possibility could be that the protein is also
shortened at its C-terminus, which contains an excess of
basic residues (Fig. 7). Indeed, t runcation of t he C-terminus
by 16–26 residues leads to a predicted IEF for CLAP which

residues after V301 (see above). In one experiment, treat-
ment of CLAP (fraction f) with PI-PLC altered the band
pattern o n an IEF gel (data not shown) suggesting that at
least one isoform terminated with a GPI unit. Overall, the
combined data indicate that the primary translation product
(prepro-CLAP) is processed at the N-terminus between G43
and E44, and probably at the C-terminus at D306, the latter
being one of the two weak sites i ndicated by the GPI
prediction tool (Discussion). A similar result would be
expected to occur during the processing of CLAP clone 2
translation product. Post-translational processing of pro-
CLAP at both th e N- and C-termini is required to achieve
the properties observed f or mature CLAP.
Fig. 6. Relative abundance of t he catalytic a nd noncatalytic su bunits of
CLP at different stages in the life cycle of Artemia. Protein extracts were
prepared from various tissues or whole organisms at different stages in
the life cycle of Artemia, then 33–135 lg were subjected to SDS/PAGE
and Western blot analyses along with five different concentrations of
purified Artemia cathepsin L in separate lanes. The solid bars represent
the noncatalytic subunit (CLAP), while the u nfilled bars represent t he
catalytic subunit (CL). EE, ovisacs containing e ncysted embryos
(33 lg protein); NEE, ovisacs containing nonencysted embryos (34 lg
protein); GF, gravid females somatic tissue (126 lg protein); NGF,
nongravid adult females (135 lg protein); M, adult males (135 lg
protein).
Ó FEBS 2004 Cathepsin L and cell adhesion protein in Artemia (Eur. J. Biochem. 271) 4019
Higher order structure of CLAP
The secondary structure o f CLAP w as predicted a ccording
to
PREDICTPROTEIN

CLAP_1:GACATGAAACTGAGAACTCTCCTCACAAAGAGGGACTTGAGGATTAATTTGTATGACAATGGGCAGACAATTCTTGCCGGTGGGAAACGTATAAATGGAT 500
CLAP_2:GACATGAAACTGAGAACTCTCCTCACAAAGAGGGACTTGAGGATTAATTTGTATGACAATGGGCAGACAATTCTTGCCGGTGGGAAACGTATAAATGGAT 432
D M K L R T L L T K R D L R I N L Y D N G Q T I L A G G K R I N G S
155
CLAP_1:CAAATTATGAAGCTCACAATGGTGTTCTGCATCTCCTTGAAGATGTGATTGTCTCTATACCAGCACGACATGGAACAGTGATTCACCAGCTGAGAAGATG 600
CLAP_2:CAAATTATGAAGCTCACAATGGTGTTCTGCATCTCCTTGAAGATGTGATTGTCTCTATACCAGCACGACATGGAACAGTGATTCACCAGCTGAGAAGATG 532
N Y E A H N G V L H L L E D V I V S
I P A R H G T V I H Q L R R C 188
CLAP_1:TCCAGTTTTTTCTGATCTTGTGGAGCTCATTGATAGAGCAGGTCTTGATGAAGCTCTTCAAACCCATGGACCTATTACTTTCTTTGCCCCAAGCAATGAT 700
CLAP_2:TCCAGTTTTTTCTGATCTTGTGGAGCTCATTGATAAAGCAGGTCTTGATGAAGCTCTTCAAACCCATGGACCTATTACTTTCTTTGCCCCAAGCAATGAT 632
P V F S D L V E L I D R A G L D E A L Q T H G P I T F F A P S N D
221
K
CLAP_1:GTCATAAGGAAACTCCCTCCTGATGTGATTAAACACCTTGTTGATGACCCAGCTCTCCTAAAAGAAGTTTTAACCTACCATGTCTTGTCTGGAACCTTCT 800
CLAP_2:GTCATAAGGAAACTCCCTCCTGATGTGATTAAACACCTTGTTGATGACCCAGCTCTCCTAAAAGAAGTTTTAACCTACCATGTCTTGTCTGGAACCTTCT 732
V I R K L P P D V I K H L V D D P A L L K E V L T Y H V L S G T F Y
255
CLAP_1:ATTCTCCTGGCATTAAAGATGGAATGGAGGGAACCACGATGCAAGGAAAGAGTCTCATATTTTCAATCAAAGATGGTGAGGTTATAATCAACAGCAAGAC 900
CLAP_2:ATTCTCCTGGCATTAAAGATGGAATGGAGGGAACCACGATGCAAGGAAAGAGTCTCATATTTTCAATCAAAGATGGTGAGGTTATAATCAACAGCAAGAC 832
S P G I K D G M E G T T M Q G K S L I F S I K D G E V I I N S K T
288
CLAP_1:TAAGGTTACCAGTGCTGATTCCAACGCATCTAATGGTGTAATTCACAGCATTGATAATGTTCTAATTCCACCACAAATTCAAGCTAAGCTGAAGCGTCGA 1000
CLAP_2:TAAGGTTACCAGTGCTGATTCCAACGCATCTAATGGTGTAATTCACAGCATTGATAATGTTCTAATTCCACCACAAATTCAAGCTAAGCTGAAGCGTCGA 932
K V T S A D S N A S N G V I H S I D N V L I P P
Q I Q A K L K R R 321
CLAP_1:ATTCTGAAGAAATCGAGAGCATTTAGCTTCCAGTAG
AAAACGGTGGTTTCGTAGTGCTTTTCTTTTCCATGGGCGTGAATGTTTCTCATTTCTCTGGTGA 1100
CLAP_2:ATTCTGAAGAAATCGAGAGCATTTAGCTTCCAGTAG
AAAACGGTGGTGTCGTAGTGCTTTTCTTTTCCATGGGCGTGAATGTTTCTCATTTCTCTGGTGA 1032
I L K K S R A F S F Q * 332

1870
Fig. 7. Nucleotide and deduced amino acid sequence of two cDNA clones coding for CLAP. Clones 1 and 2 were found to be 95% identical except for
a gap of 68 nucleotides near the 5¢-end of clone 2. Amino acid sequences determined by Edman degradation are shown in bold, and each is a perfect
match with the deduced amino acid sequence. The putative start (ATG) and stop (TAG) translation sites are double-underlined. Two fasciclin I-like
domains are underlined, and two potential N-glycosylation sites are boxed. The double-underlined and bold sequence near the 3¢-end of clones 1
and 2 (ATTAA) are putative poly(A) recognition sites. Two potential GTP binding sites are present at 199–202 (DRAG) and 265–272
(GTTMQGKS) in clones 1 and 2. Putative destabilizing elements ( AU/T-rich) in the 3¢ UTR a re underlined. The arrowheads represent putative
cleavage sites in prepro-CLAP. The asterisks represent sites in the noncoding region of clones 1 and 2 where mismatched nucleotides are present.
4020 A. H. Warner et al .(Eur. J. Biochem. 271) Ó FEBS 2004
successful [6,23,24]. In the p resent study we found that
chromatography of the CLP complex on a high perform-
ance cation matrix (Mono S) yielded both CL and CLAP in
a high s tate of purity. However, dissociation of CLP to its
subunits (CL and CLAP) required incubation of CLP at pH
5 for at least 1 h at 4 °C prior to chromatography on Mono
S. Dissociation of CLP could be blocked by inclusion of
Z-Phe-Ala-CH
2
F, a reversible cysteine protease inhibitor, in
CLP preparations, suggesting that CLAP was modified in
the process of i ts separation from CL. A ttempts to
recombine CL and CLAP into an active CLP c omplex
after purification on Mono S have not been successful. Thus
the mechanism of CLAP binding to the catalytic subunit
resulting i n CL s tabilization appears t o be c omplex and not
yet understood. CLAP might prevent ÔunzippingÕ or
destablization of the active site region of cathepsin L at
higher than normal temperature and pH as suggested for
cathepsin B [38]. The increased stability o f CL in the CLP
complex is consistent with the adaptive nature of Artemia

of E. histolytica phagocytic trophozoites to target (host)
cells such as erythrocytes, which are then consumed by
phagocytosis and degraded by the associated cathepsin L.
Cysteine proteases such as CL are used frequently by
parasitic organisms to promote i nvasion a nd destruction of
target organisms [19].
From a search of he Conserved Domain Database
(NCBI) the similarity of the t wo fasciclin domains in CLAP
with other fasciclin I containing proteins is clear (Fig. 9).
Using
BLAST
( to
identify related proteins, a putative cell adhesion protein
from the sea anemone Anthopleura elegantissima showed the
highest identity with CLAP. Other proteins of relevance
were HLC-32, a protein secreted into the extra-embryonic
matrix of sea urchins at fertilization [41], and a 30 kDa yolk
granule p rotein in sea urchins [42]. However, the sea urchin
protein self-dimerizes, while CLAP, as a component of the
Fig. 8. Structural co mparisons between
Drosophila fascicl in I and CLAP. The a mino
acid se quence of Drosophila fasciclin chain A
(NCBI database entry 1070), was aligned with
the proposed (mature) translation product, i.e.
with polypeptide 44–306, of clone 1 o f CLAP
using the
CLUSTALW
multiple alignment p ro-
gram. The d eterm ined secondary structure
(alpha helices and beta strands), as b ased on

heterodimeric CLP at the surface of yolk platelets [3],
appears to dimerize (in vivo ) only with CL.
The function of the Fas I domains in CLAP is unknown,
but generally Fas I domains are thought to represent ancient
cell adhesion domains [37]. Of importance to understanding
the structure and function of CLAP, is that most proteins
containing Fas I domains are anchored to cell membranes
through a GPI unit at the C-terminus of the protein [36].
Thus, while the GPI Predictor tool (.
univie.ac.at/sat/gpi/gpi_server.html) did not show a GPI
attachment site near the C-terminus of pro-CLAP, t he
possibility exists that mature CLAP is terminated with a
GPI unit a t N299 o r D306, w eak sites identified b y the GPI
Predictor tool. The observation that PI-PLC produced an
altered band pattern in CLAP suggests that a GPI unit is
present at the C-terminus. Addition of GPI, if it occurred,
would b e accompanied by cleavage o f the highly basic
peptide chain behind the modified residue [43]. Such a
modification of pro-CLAP would result in a predicted
molecular mass closer to that observed for mature CLAP by
SDS/PAGE (31.5 kDa), and a n i soelectric point in the
range of values observed by IEF (p I 5 .9–6.1). Processing of
the pro-CLAP C-terminus is essential to lower the mole-
cular mass and pI of the protein to values observed by SDS/
PAGE and IEF. Interestingly, pool sequencing of the
mixture o f CNBr pep tides generated from CLAP revealed
in the C-terminal CNBr peptides, the presence of N299,
G300, and V 301 i n s equence c ycles 31–3 3, with V301 being
the last visible residue of this peptide. Thus, because N299 is
observed in the C-terminus in an internal position, we infer

would result in a pro-CLAP that would avoid trafficking
through the ER/Golgi complex.
Immunocytochemical and cell fractionation methods
demonstrated that considerable amounts of CLP reside at
the surface of yolk platelets in Artemia, but that the pathway
that CLAP , CL or CLP takes to the surface of platelets is
unknown. While we can only speculate at this time about
the mechanism of C LP attachment to yolk platelets, neither
lysosomes nor transport v esicles are visible at t he surface of
mature platelets [44]. However, electron microscopy has
shown that yolk platelets acquire a vesiculated periphery
during vitellogenesis which may represent the uptake of
vesicles containing CLP derived from the ER/Golgi path-
way [ 3,44]. T he fact that yolk platelets i n s ea urchin possess
a 30 kDa fasciclin-co ntaining p rotein with a high d egree o f
homology with Artemia CLAP is noteworthy [42].
Considerable CLP has been detected in extracellular
regions of embryos a nd in tissues of l arvae, especially in the
developing gut [3]. T ransport of CLP to extracellular sites
probably requires molecular signals different from those
that direct transport of C LP to th e surface o f yolk platelets.
How this m ight occur is speculative, but it should be n oted
that the C-terminus of Artemia CL contains a secretion
signal (ASYPLV) nearly identical to signals that promote
CL secretion in mammalian tissues [24,45] and parasitic
nematodes [ 2]. Localization o f CLP in the e xtracellular
matrix could occur through the Fas I domains or putative
GPI unit, if one exists in CLAP as it does in Drosophila
fasciclin I and many other fasciclin-containing proteins
[36,46]. Fas I domains in proteins are a lmost always found

Finally, we have not yet investigated the potential
importance of the nucleotide binding domain in CLAP,
but the p resence of this domain suggests a n energy-
dependent mechanism for CLP translocation to various
sites in embryos and l arvae or for C-terminus modification
[43]. Fas I containing proteins generally lack nucleotide
Ó FEBS 2004 Cathepsin L and cell adhesion protein in Artemia (Eur. J. Biochem. 271) 4023
binding domains, so the presence of a GTP/ATP binding
region in CLAP may indicate an energy dependent mech-
anism for pro-CLAP processing or cathepsin L docking
and stabilization not found in other organisms.
Acknowledgements
The authors wish to thank the Natural Sciences and Engineering
Research Co uncil of Canada for the ir financial support of t his s tudy.
We also wish to thank Dr T homas MacRae of Dalhousie University
and Dr Dora Cavallo-Medved of Wayne State University for their
critical reading a nd comments of an ea rlier version of this work.
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