Warner1, Ervin Pullumbi1, Reinout Amons2and Liqian Liu1 1 Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada;2Department of Molecular Cell Biology, Sylviu
Trang 1Characterization of a cathepsin L-associated protein in Artemia
and its relationship to the FAS-I family of cell adhesion proteins
Alden H Warner1, Ervin Pullumbi1, Reinout Amons2and Liqian Liu1
1
Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada;2Department 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 as monomeric cathepsin L below
pH 5 The heterodimer was more stable than the monomer
between pH 6 and 7.4 and at 30–50C Artemia CLAP and
cathepsin L are present in nearly equimolar amounts at all
stages in the life cycle and most abundant in encysted eggs
and embyros Moreover, CLAP, either free or as a complex
with cathepsin L, was resistant to hydrolysis by cathepsin L
Two clones coding for CLAP were isolated from an Artemia
embryo cDNA library and sequenced Both clones have nearly identical open reading frames, but show differences at the 5¢- and 3¢-termini Each cDNA clone has an extensive 3¢-untranslated region containing 70–72% A+T The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276) This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos
Keywords: Artemia; cathepsin L; cell adhesion proteins; fasciclins
Cathepsin L (CL) is a ubiquitous cysteine protease in
eukaryotes and essential for development in several
organ-isms including Xenopus laevis [1], Caenorhabditis elegans [2],
and Artemia franciscana [3] Inhibition of CL activity in
these organisms, or deletion of the CL gene, leads to severe
abnormalities and even death Developmental events
dependent on cysteine protease activity are numerous and
include yolk utilization [3–5], activation of latent enzymes
[6], gastrulation [1], differentiation [7–9], tissue remodelling
[10], implantation [11], and molting [3,12,13] In developing
embryos, cysteine proteases are often found in the
cyto-plasm and 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 in intracellular protein turnover
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 secretion of cysteine 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 31.5 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] The 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; the 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 appearing at the surface of yolk platelets and in the extracellular matrix [3,25]
cDNAs encoding the CL subunit of Artemia CLP have been isolated and sequenced and their amino acid
Correspondence to A H Warner, Department of Biological Sciences,
University of Windsor, Windsor, Ontario, N9B 3P4, Canada.
Fax: + 519 971 3609, E-mail: warner1@uwindsor.ca
Abbreviations: CL, cathepsin L, catalytic subunit, monomer; CLP,
cathepsin L-like protease, dimer; CLAP, cathepsin L-associated
protein; PI-PLC, phosphatidylinositol-specific phospholipase C;
GPI, glycosyl-phosphatidylinositol; CNBr, cyanogen bromide;
TNBS, trinitrobenzenesulfonic acid.
(Received 23 April 2004, revised 19 July 2004,
accepted 19 August 2004)
Trang 2composition deduced [24] At the amino acid level, Artemia
CL has 73.9% identity with Drosophila CL and 68.7%
identity with human CL Despite the high degree of
similarity with Drosophila, human and other cathepsin Ls,
ArtemiaCL appears to function as a heterodimer (i.e., CLP)
of 60 kDa and not as a monomeric protein like in other
eukaryotes Until now the noncatalytic subunit of CLP (i.e.,
CLAP) has received little attention
This report focuses mainly on characterization of CLAP
and its potential role in the function of CL Herein, we
present evidence that CLAP enhances the stability of CL to
temperatures and pH normally inconsistent with CL
activity Primary sequence analysis of CLAP and cDNA
clones coding for CLAP show it to be a cell adhesion
protein and member of the fasciclin I family of proteins
These results support the hypothesis that CL in Artemia
embryos is unique and functions outside lysosomes, in the
cytoplasm and extracellular matrix, unlike CL in many
other higher eukaryotes
Materials and methods
Purification of cathepsin L-like protease
The cathepsin L-like protease (CLP) in embryos of the
brine shrimp, A franciscana was purified using a
modifica-tion of a published method [24] Fifty grams of fully
hydrated Artemia cysts were homogenized in ice-cold
homogenization buffer (50 mM Tris/HCl, pH 7.2, 5 mM
KCl, 1 mM dithiothreitol and 10 mM MgCl2) using a
motorized mortar and pestle (Torsion Balance Co, Clinton,
NJ, USA) 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 to obtain the 35–75% ammonium
sulfate insoluble material The latter was collected by
centrifugation, dissolved in Buffer A [15 mM potassium
phosphate, pH 6.8, 25 mM KCl and 10% (w/v) glycerol],
then desalted on a column of Sephadex G-25 using Buffer A
as the eluent The protease was purified to near
homo-geneity by sequential chromatography on
DEAE–Seph-arose, Concanavalin A–SephDEAE–Seph-arose, Superose 12 and Mono
Q [23,24] The major isoforms of Artemia CLP that eluted
from the Mono Q column were combined and concentrated
to about 1 mL using Centricon 10 filters (Amicon Canada,
Oakville, ON, Canada) All chromatographic media were
from Amersham Pharmacia Biotech (Baie d’Urfe, QC,
Canada)
Protein and protease assays
The protein content of all column fractions was determined
by the Bio-Rad microassay [26] or bicinchoninic acid assay
[27] using BSA as the protein standard Cysteine protease
activity of column fractions was determined using protamine
sulfate as substrate and the trinitrobenzene sulfonic acid
(TNBS) method [23] One unit of protease activity was
defined as the release of 1 micromole of amino peptide per
minute from the substrate at pH 4.0 and 40C CL assays
were carried out using a modified method of Barrett &
Kirschke [28] All reaction vessels contained the following:
0.2 m Cbz-Phe-Arg-4-methoxy-b-naphthylamide, 83 m
potassium phosphate, pH 5.0, 0.67 mM EDTA, 0.5 mM
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 mMmersalyl acid, 30 mMNaOH, 2% (v/v) Brij and 0.81 mMEDTA, adjusted to pH 4.0 with 1M
HCl] to which was added an additional volume of coupling buffer containing 0.5 mgÆmL)1Fast Garnet (Sigma, Mis-sissauga, ON, Canada) After 15 min incubation at room temperature, the complex was extracted with 1 mL n-butanol and the color intensity determined by analysis at 520 nm The number of pmoles of cathepsin L were determined by titration of the active 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 pH 5.0 and temperature indi-cated Artemia p26 protein was a gift of T MacRae (Dalhousie University, Halifax, NS, Canada), while the protein artemin was prepared from Artemia cysts [30]
Isoelectric focussing and sodium dodecylsulfate polyacrylamide gel electrophoresis
Isoelectric focussing (IEF) was performed in glass tubes (0.5· 12 cm) containing 6% (w/v) acrylamide, 2% (v/v) 4/6 ampholytes (Bio-Lyte; Bio-Rad, Mississauga, ON, Canada), 1% (v/v) 3/10 ampholytes (Bio-Lyte), and 12.5% (v/v) glycerol using a Haake–Buchler unit (Baxter, McG-raw Park, IL, USA) The protein samples contained 10% (v/v) glycerol, 0.1% (v/v) 3/10 ampholyte, 0.002% (w/v) bromphenol blue and either CLAP or IEF standards (pI 4.45–9.6) in a final volume of 0.1 mL The top buffer (catholyte) was 100 mM NaOH and the bottom buffer (anolyte) was 3 mMindole-acetic acid Isoelectric focussing was initiated at 350 V and 1.5 mA per gel column, and the focussing was completed by 18 h at 4C The ampholytes and IEF standards were from Bio-Rad Following electro-phoresis, the gels were soaked in several changes of distilled water for about 10 min then stained with the Bio-Rad silver reagent as recommended by the supplier A control gel containing buffer in place of protein was washed briefly
in distilled water, then 0.5 cm sections were placed in 1.0 mL distilled water for pH measurement Gels contain-ing the IEF standards and buffer only gave identical linear responses with gel length In a separate experiment, CLAP 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) methanol 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
Trang 3Cysteine protease analysis at different stages
in theArtemia 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 N2 Gravid females from which the ovisacs
had been removed were saved for analysis Immature,
nongravid females containing no visible signs of eggs, and
adult males, were collected, washed and frozen in liquid N2
All tissues were stored at) 70 C until needed The frozen
tissues were homogenized in a buffer containing 50 mM
sodium phosphate, pH 7.4, 1 mM EDTA and 5% (w/v)
SDS (at 70C) using small glass homogenizers The
insoluble material was removed by centrifugation, and
aliquots were taken for protein measurement and analysis in
7–18% SDS/PAGE gels The amounts of catalytic and
noncatalytic subunits of CLP in each tissue extract were
determined by densitometry as described previously [25]
Amino acid sequencing of CLAP and CLAP fragments
Mono S purified and untreated CLAP was subjected to
Edman sequencing on a Hewlett–Packard G1005A
pro-tein sequencer A cyanogen bromide (CNBr) generated
peptide of CLAP of about 25 kDa was purified by SDS/
PAGE, transferred to a poly(vinylidene difluoride)
mem-brane and sequenced by Edman degradation along with
five peptides obtained by Lys-C treatment of CLAP
(Eastern Quebec Peptide Sequencing Facility, Ste-Foy,
QC, Canada) In addition, pool sequencing, i.e
sequen-cing of the complete mixture of CNBr-generated peptides,
was also performed
Isolation and sequencing of cDNA clones encoding CLAP
A cDNA library prepared from cysts of A franciscana was
a gift from T MacRae (Dalhousie University, Halifax, NS,
Canada) prepared originally by L Sastre (Instituto de
Investigaciones Biome´dicas, CSIC/UAM, Madrid, Spain)
The library was constructed in phage kZAP II (Stratagene,
La Jolla, CA, USA) with the cDNAs were 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 a32P-labeled 564 bp PCR
product generated using primers constructed from amino
acid sequence data of CLAP, and cloned into pCR2.1
(Invitrogen, Burlington, ON, Canada) Approximately
2· 106 plaques were screened using standard protocols
[34], and six plaques, identified by hybridization to the
564 bp PCR product, were chosen for further analysis
The isolated phage were converted to Bluescript
phage-mids using ExAssist helper phage and a protocol provided
by the supplier (Stratagene) Six cDNA clones were grown
overnight in the presence of ampicillin (50 lgÆmL)1) and the
DNA was isolated using the Wizard Miniprep Kit (Promega,
Madison, WI, USA) All clones showed identical restriction
maps, and two were sequenced by cycle sequencing using
primers constructed from the original 564 bp PCR product
and from information in the Bluescript phagemid Sequen-cing was performed on a Visible Genetics (Suwanee, GA, USA) instrument using the Thermo Sequenase Cy5.5 Terminator Cycle Sequencing Kit (Amersham Pharmacia) Results
Separation ofArtemia CLP subunits by HPLC on Mono S Various fractionation methods have been attempted to separate the catalytic and noncatalytic subunits of Artemia CLP without loss of protease activity, but none has been successful except cation-exchange chromatography How-ever, partial separation of Artemia CLP subunits was achieved by chromatography on Mono S following pre-incubation of the complex at pH 5 for at least 2.5 h at 0–4C, including dialysis (Fig 1A) This step resulted in two, partially separated, fractions of CLAP (e and f) which could not be resolved completely by re-chromatography on Mono S (Fig 1B,C)
Fig 1 Fractionation of Artemia cathepsin L subunits by HPLC on a Mono S column Prior to chromatography on Mono S 0.9 mg of purified heterodimeric Artemia cathepsin L was adjusted to pH 5.0 with 1 M sodium acetate, pH 4, incubated for 1 h at 0 C, then dia-lyzed against Buffer A (A) Elution profile of the Artemia cathepsin L subunits monitored at 280 nm and expressed in mV on the y axis Column fractions in the region of a–f were concentrated for protease assays and subunit analysis by SDS/PAGE (Fig 2A,B) (B,C) Frac-tions e and f were re-chromatographed on the Mono S column under conditions identical to those used initially.
Trang 4The 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 of 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 ofArtemia CLAP by isoelectric focussing
CLAP fractions e and f (Fig 1B,C) were analyzed by IEF
Fractions e and f showed three and four bands, respectively,
on IEF gels with pI values ranging from 5.9 to 6.1 (Fig 3)
Fractions e and f have at least one unique isoform each (pI 5.9 for e and pI 6.1 for f), while two bands of pI 5.95 and pI 6.0 were common to each of the major CLAP fractions, although this does not mean that these are identical isoforms Overall, Artemia CLAP appears to contain four isoforms in nearly equal amounts, but these isoforms were not resolved by chromatography on a C-18 reverse phase column in which fractions e and f showed identical elution characteristics using acetonitrile/trifluoroacetic acid as the eluent (data not shown and [24])
Activity of dimer and monomer forms ofArtemia CLP
at different pH and temperatures Freshly prepared Artemia CLP (60 kDa, dimer) and CL (28.5 kDa, monomer) (Fig 1A, peak a) were assayed for
CL activity in parallel reaction vessels at 30C and various
pH (Fig 4A) The monomer showed maximum activity at
pH 5.0, while the dimer showed a slightly different activity profile with the maximum around pH 4.7 The rate constants for CLP (dimer) and CL (monomer) were similar between pH 5.0 and 6.5, whereas the dimer had about
A
B
Fig 2 SDS/PAGE analysis of Artemia cathepsin L fractions from
Mono S column (A) Approximately 4.5 lg of Mono S fractions a–f
shown in Fig 1 were applied to individual lanes of a 12%
polyacryl-amide gel, and following electrophoresis, the gel was stained with
Coomassie blue The protease activity of fractions a–f was determined
prior to electrophoresis using the TNBS assay, and the results
(pro-tease activity per mg protein) are shown in brackets below each lane.
The migration position of CL and CLAP, the catalytic and
noncata-lytic subunits, respectively, of the protease are shown on the right,
while protein standards are shown on the left (B) Lanes labelled e (1.5
and 3.0 lg) and f (1.5 and 3.0 lg) show the electrophoretic position of
CLAP fractions e and f, respectively, after re-chromatography on
Mono S (Fig 1B,C) The (0) at the bottom shows the absence of
protease activity in e and f after re-chromatography Mw, molecular
mass marker.
Fig 3 Isoelectric focusing of CLAP Twenty-five micrograms of CLAP fractions e and f (Fig 1B,C) in a volume of 100 lL were applied to the top of separate glass tubes containing 6% acrylamide as described in Materials and methods 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 both IEF standards (Std) and buffer control gel run in parallel The numbers at 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 protein based on its deduced amino acid composition.
Trang 52-fold higher activity at pH 4.3–4.7 Preincubation (1 h at
30C) 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 40C 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 all incubation temperatures between 40 and 53C (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 Artemia’snatural environment [6]
Resistance of CLAP to degradation byArtemia cathepsin L monomer
Early research on the Artemia cysteine protease demonstra-ted that native CLP undergoes autodegradation when stored below pH 5 irrespective of temperature [23] In the present study we tested the sensitivity of CLAP and BSA, artemin, and p26 to the Artemia CL Results showed that CLAP is resistant to hydrolysis by CL at 30C and pH 5.0, while BSA and two abundant proteins in Artemia embryos, artemin and p26, are degraded by Artemia CL after 60 min incubation (Fig 5)
Abundance of the catalytic and noncatalytic subunits
of CLP at various stages in theArtemia life cycle Artemiagrown in the laboratory were collected at different stages in the life cycle, and total protein isolated from different tissues or whole animals was analyzed for the catalytic and noncatalytic subunits using Western blotting after SDS/PAGE separation of the proteins Ovisacs with encysted embryos contained the largest amount of both protease subunits (about 0.15% of total protein) in nearly equimolar amounts (Fig 6) Ovisacs containing nonency-sted embryos contained considerably less of the Artemia CLP subunits (0.038% of the total protein in the extract), while somatic tissues in gravid females and immature females had still smaller amounts of each subunit
Fig 4 Activity of the monomeric and dimeric forms of Artemia embryo
cathepsin L at different pH and temperatures (A) CLP (dimer) and CL
(monomer) were assayed at different pH for cathepsin L activity (rate
constants) Each reaction vessel contained 40–60 pmoles of the active
protease (B) Different forms of the protease (solid bars, CLP; unfilled
bars, CL) were incubated for 1 h at 30 C in 25 m M KCl, 10 m M
sodium phosphate, 10% glycerol and 0.2 mgÆmL)1BSA at the pH
indicated, then assayed for cathepsin L activity at pH 5.0 and 30 C
and the rate constants determined The control represents CL
(monomer) and CLP (dimer) maintained at 0 C and pH 6.8 prior to
the assay (C) Incubation vessels were set up to contain 80–100 pmoles
of CL (monomer) and CLP (dimer) 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 for cathepsin L activity at pH 5.0, and their
rate constants determined.
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 volume of 40 lL After 0 and 60 min incu-bation at 30 C, 10 lL were taken from each reaction vessel 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 proteins with their molecular mass (kDa) shown at the left The migration position of the Artemia cathepsin L monomer is shown at the right (ACL) Faint bands at 16–18 kDa in the 60 min lanes rep-resent CL autodegradation products observed in similar experiments using Western blotting.
Trang 6( 0.01%) Adult males had the lowest level (< 0.01%) of
CL and CLAP of any tissue tested
Amino acid sequence of cathepsin L-associated protein
Early attempts to sequence CLAP by Edman degradation
yielded no results, suggesting that the N-terminus of the
protein was blocked However, amino acid sequence was
obtained from a 25 kDa fragment generated by CNBr
treatment of CLAP (DNVIDHEGKFTLFAPTNEAF),
and from a peptide (KSLIFSIK) generated by Lys-C
treatment of CLAP More recently, we obtained the
sequence EAKNLVDLAESLGLSILVKALE from Edman
degradation of an untreated preparation of CLAP
indica-ting that the N-terminus of the mature protein begins with
a glutamic acid residue To obtain the full amino acid
sequence of CLAP, an Artemia cDNA library in phage
kZAP II was screened with a PCR derived probe and six
clones potentially coding for the CLAP were isolated
Following excision of Bluescript phagemid from kZAP II,
two cloned cDNAs were amplified and sequenced (Fig 7)
Clone 1 contained 1888 nucleotides with two potential start
codons (nucleotides 38–40 and 92–94) and an open reading
frame of 996 nucleotides Clone 2 contained 1870
nucleo-tides with one potential start codon (nucleonucleo-tides 24–26) and
an open reading frame spanning 945 nucleotides Clone 2
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 to K Both 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 clones 1 and 2 are composed of about 72% A + T and differed from each other by 2.1% Also, clone 1 contains seven consensus AT-rich motifs, while clone 2 contains five AT-rich motifs Both clones contain several putative poly(A) addition signals (AAT AAA and ATTAA) The nucleotide sequences of clone 1 and 2 have been entered into the NCBI database with accession numbers AY307377 and AY462276, respectively 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 mature protein terminated 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, truncation of the C-terminus
by 16–26 residues leads to a predicted IEF for CLAP which fits the observed data nicely The conclusion that part of the C-terminus is indeed missing is also supported by direct amino acid sequence analysis of CLAP being cleaved by CNBr because we could follow the sequences of all five CNBr peptides expected (not shown), beginning with E44, D70, K124, E264 and Q269, respectively The C-terminal CNBr peptide beginning with Q269 could be followed until V301 (in cycle 33), suggesting that the C-terminus of the deduced protein has been truncated at or a few residues after V301 (see below)
A high stringency search of the NCBI Conserved Domain Database (http://www.ncbi.nlm.nih.gov/) revealed two domains in Artemia CLAP with a high degree of homology with fasciclin I, an extracellular protein found in numerous organisms The first Fas I domain in clone 1 spans 128 amino acids (45–173), while the second Fas domain spans
132 amino acids (177–309) (Fig 7) Analysis of CLAP cDNA (clone 1) using EXPASY revealed potential GTP binding sites at positions 99–202 (DRAG) and 265–272 (GTTMQGKS) [35] Having recognized CLAP as a mem-ber of the fasciclin family of proteins, we were interested to know whether the protein – like many other fasciclins – has been modified C-terminally with a GPI moiety [36] The presence of such a moiety would possibly account in part for the protein’s observed heterogeneity when analyzed by IEF Moreover, the truncation of the protein chain beyond the site of GPI modification would be in line with the pool sequencing results, which suggest only a few additional residues after V301 (see above) In one experiment, treat-ment of CLAP (fraction f) with PI-PLC altered the band pattern on 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 indicated 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 the N- and C-termini is required to achieve the properties observed for mature CLAP
Fig 6 Relative abundance of the catalytic and noncatalytic subunits 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 unfilled bars represent the
catalytic subunit (CL) EE, ovisacs containing encysted 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).
Trang 7Higher order structure of CLAP
The secondary structure of CLAP was predicted according
toPREDICTPROTEINavailable at Columbia University
Bioin-formatics Center (http://www.predictprotein.org/) (Fig 8)
For comparison the same figure includes the secondary
structure of chain A, a fasciclin I domain of Drosophila
melanogaster, derived from its observed spatial structure
[37] Both proteins were aligned using the CLUSTALW
program; they share substantial amounts of secondary
structural elements, and they are clearly related to each other regarding their amino acid sequences (Fig 9) Whether this correspondence points to similar roles in extracellular function of both proteins remains to be seen although they probably have a common evolutionary origin
Discussion Previous attempts to obtain Artemia cathepsin L-associated protein (CLAP) in an undenatured form had not been
CLAP_1:AATTCGGCACGAGGCAAAAACAAATAAATGCTTAATTATGTTGTATATTATTCCATTATTTCTTATTATTGGCTGCTCAAATGCCATATGGATGTTAAAT 100 CLAP_2:AATTCGGCACGAGG -GCCATATGGATGTTAAAT 32
M L Y I I P L F L I I G C S N A I W M L N 21
CLAP_1:TTGAATGCTGTCACCACTGAGCCAGAAGCTAAGCTAGAACATGCTGCTATCCCTATCAAAGATGGTGAGGCAAAAAACCTTGTGGATCTTGCAGAGTCTC 200 CLAP_2:TTGAATGCTGTCACCACTGAGCCAGAAGCTAAGCTAGAACATGCTGCTATCCCTATCAAAGATGGTGAGGCAAAAAACCTTGTGGATCTTGCAGAGTCTC 132
L N A V T T E P E A K L E H A A I P I K D G E A K N L V D L A E S L 55
CLAP_1:TTGGACTGTCCATCCTTGTCAAGGCTCTTGAAGAAACTGGAATGGATAATGTGATTGATCATGAAGGTAAATTTACTTTATTTGCTCCAACTAATGAAGC 300 CLAP_2:TTGGACTGTCCATCCTTGTCAAGGCTCTTGAAGAAACTGGAATGGATAATGTGATTGATCATGAAGGTAAATTTACTTTATTTGCTCCAACTAATGAAGC 232
G L S I L V K A L E E T G M D N V I D H E G K F T L F A P T N E A 88
CLAP_1:ATTTAAAAGAATTCCCGAATGGGCCAAGGATCTTCCATTGAAAGAAGTTTTGAGGTATCACATTGCAAGAGGGTTGTATTATGATAAAGATCTCCAGAAT 400 CLAP_2:ATTTAAAAGAATTCCCGAATGGGCCAAGGATCTTCCATTGAAAGAAGTTTTGAGGTATCACATTGCAAGAGGGTTGTATTATGATAAAGATCTCCAGAAT 332
F K R I P E W A K D L P L K E V L R Y H I A R G L Y Y D K D L Q N 121
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:ATTCTGAAGAAATCGAGAGCATTTAGCTTCCAGTAGAAAACGGTGGTTTCGTAGTGCTTTTCTTTTCCATGGGCGTGAATGTTTCTCATTTCTCTGGTGA 1100 CLAP_2:ATTCTGAAGAAATCGAGAGCATTTAGCTTCCAGTAGAAAACGGTGGTGTCGTAGTGCTTTTCTTTTCCATGGGCGTGAATGTTTCTCATTTCTCTGGTGA 1032
I L K K S R A F S F Q * 332
CLAP_1:AAGTCTGTCGTCAAAATGTTATGAACGTCTCTTGTCATAAAGAAAGATAACCTCTCTTTTTAGTTTGGTTTAGATATTAAGGACAGATCCAAAATATTTG 1200 CLAP_2:AAGTCTGTCGTCAAAATGTTATGAACGTCTCTTGTCATAAAGAAAGAGAACCTCTCTTTTTAGTTTGGTTTAGATATTAAGGACAGATCCAAAATATTTG 1132
*
CLAP_1:AGGACCTTTTATTAGACATTTCAAATATATAATAAACGTTATTTTAAAATTAGAAAAATTGAAAGACAAGCTAATGAAAGCTTATTGCCGATTGGAAAGT 1300 CLAP_2:AGGACCTTTTATTAGACATTTCAAATATATAATAAACGTTATTTTAAAATTAGAAAAATTGAAAGACAAGCTAATGAAAGCTTATTGCCGATTGGAAAGT 1232
CLAP_1:TTGCTTGGGGGGAAGACTCGTTACAATTCTTTTTCTTTATTTTCTTTTTAGGTAGCTTCTTTATTTTATTTTTTT-ATCTCTTTCTTGATTTTCTTTTCT 1399 CLAP_2:TTGCTTGGGTG-AAGACTCGTTACAATTCTTTTTCTTTATTTTCTTTTTAGGTAGCTTCTTTATTTTATTTTTTTTATCTCTTTCTTGATTTTCTTTTCT 1331
* * *
CLAP_1:GGCAACTTCTTTATATTTTTCTTATTTCTGTTCTTTATTTCTTTATTTTTTGAATAGTTTCTATTGCTATAGGATTAGCTTGTCTAAGTAAATTCTAAGT 1499 CLAP_2:GGCAGCTTCTTTATATTTTTCTTATTTCTGTTCTTTATTTCTTTATTTTTTGAATAGTTTCTATTGCTATAGGATTAGCTTGTCTAAGTAAATTCTAAGT 1431
*
CLAP_1:TTTTTTTTTTTTTTAATCAGAAAAACACTAGATTTCGTAAGATTAATGTGGGTTTCATGAAAACCTTTTTATTGACATT-TAAATAAATTGGGTTTTGCA 1598 CLAP_2:TTTTTTTTTTTTTAAATCAGAAAAACACTAGATTTCGTAAGATTAATGTGGGTTTCATGAAAACCTTTTTATTGACATTCTAAATAAATTGGGTTTTGCA 1531
* *
CLAP_1:CAAGTTTCTTGGACTTTA-GAAAAGTATGTTTAATTTTTCATAAGAATGTCTAAGGTTTCGTATTTTTTTACACAAATACTTCAACCGAGAGGATTCCAT 1697 CLAP_2:CAAGTTTCTTAGACTTTAAGAAAAGTATGTTTAATTTTTCATAAGAATGTCTAAGGTTTCGTAATTGTT-ACACAAATACTTCAATCGAGAGGACTCCGT 1630
* * * * * * * *
CLAP_1:ATTAGTGCTATAGTTTGGGAAATATTTAGCCCTTGTTTTGTGTGATCTTATAAGATAATATTTGTAGTTTGTGCTTTTATATAATTTAGCTCATTGGATT 1797 CLAP_2:ATTAGTGCTATAGTTTGGGAAATATTTAGTCCTTGTTTTGTGTGATCTTATAAGATAATATTTGTAGTTTGTGCTTTTATATAATTTAGCTCATTGGATT 1730
*
CLAP_1:AAGATCTTCTGAATGTGATTATATGCGGCTGTGTTTTCTAATAGATTTCTAGATACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1888
CLAP_2:AAGATCTTCTGAATGTGATTATATGCGGCTGTGTTTTCTAATAGATTTCTAGATACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(A)48 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 are 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.
Trang 8successful [6,23,24] In the present 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 state 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 4C prior to chromatography on Mono
S Dissociation of CLP could be blocked by inclusion of
Z-Phe-Ala-CH2F, a reversible cysteine protease inhibitor, in
CLP preparations, suggesting that CLAP was modified in
the process of its separation from CL Attempts to
recombine CL and CLAP into an active CLP complex
after purification on Mono S have not been successful Thus
the mechanism of CLAP binding to the catalytic subunit
resulting in CL stabilization appears to be complex and not
yet understood CLAP might prevent unzipping 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 of CL in the CLP
complex is consistent with the adaptive nature of Artemia
embryos which have the ability to survive harsh
environ-mental conditions [39]
Analyses of CLAP on IEF gels revealed four isoforms
with pI values ranging from 5.9 to 6.1 Staining for
phosphate adducts on the Artemia CLP (heterodimer) and
purified CLAP were negative (data not shown) The
presence of N-linked carbohydrates in CLAP demonstrated
previously could generate CLAP isoforms [24], but this idea
needs further experimentation Another possible reason for
the isoforms is that the C-terminus of mature CLAP
contains heterogeneous GPI units resulting from a
post-translational event as discussed below
As demonstrated in this study the activity of CLP and CL
was similar between pH 5 and 6.5, while CLP showed about
twofold greater activity below pH 5 CLP was also more stable than CL near neutral pH and 30–40C Addition of purified CLAP, in equimolar amounts, to reaction mixtures containing CL did not affect the protease activity at pH 4, 5
or 6 Reasons for these observations are not clear, but they may be due to modifications in CLAP during incubation of CLP at pH 5 prior to chromatography on Mono S The fact that we have not been able to achieve recombination of CL and CLAP to form the naturally occurring CLP complex
in vitrois consistent with the latter observation
A search of the literature has revealed that heterodimeric CLP in Artemia is functionally similar to a novel cysteine protease in Entamoeba histolytica known as adhesin [40] Adhesin is a heterodimer composed of a cathepsin L-like protease and a protein with an adherence domain contain-ing four glycosylation sites Adhesin promotes the bindcontain-ing
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 invasion and destruction of target organisms [19]
From a search of he Conserved Domain Database (NCBI) the similarity of the two fasciclin domains in CLAP with other fasciclin I containing proteins is clear (Fig 9) Using BLAST (http://www.ncbi.nlm.nih.gov/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 protein in sea urchins [42] However, the sea urchin protein self-dimerizes, while CLAP, as a component of the
Fig 8 Structural comparisons between
Drosophila fasciclin I and CLAP The amino
acid sequence 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 of CLAP
using the CLUSTALW multiple alignment
pro-gram The determined secondary structure
(alpha helices and beta strands), as based on
the spatial structure of the Drosophila protein
[37], is indicated by single and double
under-lining, respectively The predicted secondary
structure of CLAP according to
PREDICT-PROTEIN is presented in the same way.
Trang 9β
β
β
Fig 9 Comparison of the fasciclin I domains in CLAP with selected proteins in the protein database The Conserved Domain Database of NCBI was screened with the protein coding sequence of clone 1 of CLAP and the Fas I domains in the protein compared with 10 other fasciclin I-containing proteins The most highly conserved sequences (containing more than four amino acids) are boxed, and the number below each highly conserved sequence indicates the percent identity to Artemia CLAP with the consensus sequence for each region of the fasciclin domains Because the fasciclin domains in the Conserved Domain Database are compared with only one of the four domains present in the Drosophila protein [37], its sequence 501–616 appears twice in the figure In addition, it should be noted that the alignment between Artemia and Drosophila proteins also differs from Fig 8, because, in the latter figure, a different computer program ( CLUSTALW ) was used Details of each sequence above can be found in the NCBI protein sequence database as follows: Art-clap1 (Artemia cathepsin L-associated protein, clone 1, AAP69998), Dros-fasI (Drosophila fasciclin I, NP_732166), Ory-big-H3 (Oryctolagus, rabbit, transforming growth factor-b induced protein precursor, Q95215), Homo-osf2 (Homo sapiens, osteoblast specific factor 2, S36111), Antho-cap (Anthopleura elegantissima putative cell adhesion protein Sym32, AAF65308), Scoel-lipo (Strep-tomyces coelicolor A3 putative lipoprotein, NP_624948), Rdur-osf2 (Deinococcus radiodurans osteoblast specific factor-2 related protein, NP_294122), Lyt-30kDaYP (Lytechinus variegatus 30 kDa yolk granule protein, AAG02421), Mus-tgf-bi (Mus musculus transforming growth factor-b induced protein IG-H3 precursor, Q95215), Mus-osf2 (Mus musculus osteoblast specific factor-2 pending protein, AAH31449),
Smel-ind-pr (Sinorhizobium meliloti Nex 18 symbiotically induced conserved Smel-ind-protein, NP_435828), and Syn-hypo-pp (Synechocystis sp hypothetical Smel-ind-protein s111483 precursor, P74615).
Trang 10heterodimeric 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 (http://mendel.imp
univie.ac.at/sat/gpi/gpi_server.html) did not show a GPI
attachment site near the C-terminus of pro-CLAP, the
possibility exists that mature CLAP is terminated with a
GPI unit at N299 or D306, weak sites identified by 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 be accompanied by cleavage of 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 an isoelectric point in the
range of values observed by IEF (pI 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 of CNBr peptides generated from CLAP revealed
in the C-terminal CNBr peptides, the presence of N299,
G300, and V301 in sequence cycles 31–33, 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
that the other candidate, D306, is the target for GPI
modification and site of C-terminus truncation
In some systems, the addition of GPI to the C-terminus of
a protein is an energy dependent process requiring ATP
and/or GTP [43] The fact that CLAP contains an intrinsic
ATP/GTP binding site near its C-terminus might support
this type of post-translational modification The presence of
GPI at the C-terminus of CLAP provides a mechanism to
anchor heterodimeric CLP at various sites where it is found
in embryos and newly hatched larvae [3]
Previous analysis of cDNA clones coding for
cathep-sin L, and the sequence of clone 1 coding for CLAP from
the first AUG codon onward, indicate that the
prepro-form of both CL and CLAP have well defined
hydro-phobic signals in their N-terminus (Fig 7) [24] Thus
prepro-CL and prepro-CLAP probably enter the
endo-plasmic reticulum where post-translational modifications
occur The assembly of CL and CLAP to form the
heterodimer could also occur in the ER, although the
bonds or motifs linking the two subunits have not been
determined Modifications to the predicted amino acid
sequence of prepro-CLAP, including removal of N- and
C-terminal peptides, would probably be achieved along the
ER/Golgi pathway Alternatively, prepro-CLAP could
avoid the ER by using an alternate start codon in the
mRNA (positions 92–94, clone 1), but this is unlikely as
both the N- and C-termini of prepro-CLAP must undergo
post-translational modifications that are normally
accom-plished along the ER/Golgi pathway However, translation
of clone 2 from the first start codon (positions 22–24)
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 CLP attachment to yolk platelets, neither lysosomes nor transport vesicles are visible at the 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] The fact that yolk platelets in sea urchin possess
a 30 kDa fasciclin-containing protein with a high degree of homology with Artemia CLAP is noteworthy [42] Considerable CLP has been detected in extracellular regions of embryos and in tissues of larvae, especially in the developing gut [3] Transport of CLP to extracellular sites probably requires molecular signals different from those that direct transport of CLP to the surface of yolk platelets How this might occur is speculative, but it should be noted 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 of CLP in the extracellular 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 almost always found
in the extracellular compartment of tissues, where they are believed to promote intermolecular and homotypic adhe-sion Thus CLAP, through its Fas I domains, may promote docking and stabilization of CL at various extracellular sites The resistance of mature CLAP to destruction by CL and serine proteases appearing in third and fourth instar larvae of Artemia suggests that CLAP plays an important role in CL stability and localization outside lysosomes during embryonic and early larval development [12] Analysis of the cDNA clones coding for CLAP indicated that each clone has an extensive, but slightly different 3¢ UTRs rich in AT-residues, representing AU-rich regions in CLAP mRNA AU-rich sequences in eukaryotic mRNA are thought to represent destabilizing elements leading to rapid deadenylation and messenger breakdown [47,48] Thus, while CLAP appears to be somewhat refractory to protease degradation, its mRNAs may be degraded rapidly due to AU-rich sequences in their 3¢-UTR Preliminary evidence from our laboratory on CLAP mRNA levels in developing embryos and larvae of Artemia supports the view that CLAP mRNA is unstable during development In addition, we have observed that the 3¢ UTR of CLAP mRNA contains over 125 transcription factor binding sites
as determined by the molecular toolMATINSPECTOR(http:// www.genomatrix.de/) [49] Whether these sites participate
in formation of a functional promoter for transcription of the CLAP gene or in transcription regulation of down-stream genes remains to be determined
Finally, we have not yet investigated the potential importance of the nucleotide binding domain in CLAP, but the presence of this domain suggests an energy-dependent mechanism for CLP translocation to various sites in embryos and larvae or for C-terminus modification [43] Fas I containing proteins generally lack nucleotide