ORIGINAL ARTICLE

Chlapsin, a chloroplastidial aspartic proteinasefrom the green algae Chlamydomonas reinhardtii

Carla Malaquias Almeida • Claudia Pereira •

Diana Soares da Costa • Susana Pereira •

Jose Pissarra • Isaura Simoes • Carlos Faro

Received: 23 November 2011 / Accepted: 26 January 2012 / Published online: 19 February 2012

� Springer-Verlag 2012

Abstract Aspartic proteinases have been extensively

characterized in land plants but up to now no evidences for

their presence in green algae group have yet been reported

in literature. Here we report on the identification of the first

(and only) typical aspartic proteinase from Chlamydo-

monas reinhardtii. This enzyme, named chlapsin, was

shown to maintain the primary structure organization of

typical plant aspartic proteinases but comprising distinct

features, such as similar catalytic motifs DTG/DTG

resembling those from animal and microbial counterparts,

and an unprecedentedly longer plant specific insert domain

with an extra segment of 80 amino acids, rich in alanine

residues. Our results also demonstrated that chlapsin

accumulates in Chlamydomonas chloroplast bringing this

new enzyme to a level of uniqueness among typical plant

aspartic proteinases. Chlapsin was successfully expressed

in Escherichia coli and it displayed the characteristic

enzymatic properties of typical aspartic proteinases, like

optimum activity at acidic pH and complete inhibition by

pepstatin A. Another difference to plant aspartic protein-

ases emerged as chlapsin was produced in an active form

without its putative prosegment domain. Moreover,

recombinant chlapsin showed a restricted enzymatic spec-

ificity and a proteolytic activity influenced by the presence

of redox agents and nucleotides, further differentiating it

from typical plant aspartic proteinases and anticipating a

more specialized/regulated function for this Chlamydo-

monas enzyme. Taken together, our results revealed a

pattern of complexity for typical plant aspartic proteinases

in what concerns sequence features, localization and bio-

chemical properties, raising new questions on the evolution

and function of this vast group of plant enzymes.

Keywords Aspartic proteinase � Chlamydomonas �Chloroplast � Plant specific insert

Abbreviations

AP Aspartic proteinase

PSI Plant specific insert

CDR1 Constitutive disease resistance 1

EST Expressed sequence tag

EDANS 5-((2-Aminoethyl)amino)naphthalene-1-

sulfonic acid

DABCYL 4-(Dimethylaminoazo)benzene-4-carboxylic

acid

E-64 L-trans-Epoxysuccinylleucylamide-(4-

guanidino)butane

IPTG Isopropyl b-D-1-hiogalactopyranoside

MCA (7-Methoxycoumarin-4-yl)acetyl

DNP 2,4-dinitrophenyl

rchlapsin Recombinant chlapsin

C. M. Almeida � I. Simoes � C. Faro (&)

Biocant, Biotechnology Innovation Center,

Molecular Biotechnology Unit, Parque Tecnologico de

Cantanhede, Nucleo 4 Lote 3, 3060-197 Cantanhede, Portugal

e-mail: cfaro@biocant.pt

C. Pereira � S. Pereira � J. Pissarra

Departamento de Biologia, Faculdade de Ciencias,

BioFig-Centre for Biodiversity, Functional and Integrative

Genomics, Universidade Do Porto, Rua do Campo Alegre, s/n8,4169-007 Porto, Portugal

D. S. da Costa

Biomaterials, Biodegradables and Biomimetics Research Group,

Department of Polymer Engineering, Universidade do Minho,

S. Claudio do Barco, 4806-909 Caldas das Taipas, Portugal

I. Simoes � C. Faro

CNC-Center for Neuroscience and Cell Biology of Coimbra,

University of Coimbra, 3004-517 Coimbra, Portugal

123

Planta (2012) 236:283–296

DOI 10.1007/s00425-012-1605-2

Introduction

Aspartic proteinases (EC.3.4.23) (APs) are one of the six

mechanistic classes of proteolytic enzymes and are widely

distributed in several groups of organisms like plants,

animals, bacteria, fungi and virus (Davies 1990; Simoes

and Faro 2004; Rawlings and Bateman 2009). These pro-

teases share several common features including a con-

served three-dimensional structure, consisting of two lobes

separated by a catalytic cleft where the two hallmark active

sequence motifs D-T/S-G are located. Additionally, most

of these enzymes require an acidic pH for optimal enzy-

matic activity which are specifically inhibited by pepstatin

A, and preferentially cleave peptide bonds between amino

acids with bulky hydrophobic side chains (Dunn 2002).

Most plant APs are vacuolar enzymes, involved in storage

proteins mobilization, accumulating either in the lytic

vacuole or in the protein storage vacuole (Runeberg-Roos

et al. 1994; Ramalho-Santos et al. 1998; Schaaf et al. 2004;

Pissarra et al. 2007; Pereira et al. 2008) or are secreted to

the cell wall (Vieira et al. 2001; da Costa et al. 2010). The

major differences among members of this protease family

are related with specificity/catalytic properties and with

their cellular localization which therefore results in dif-

ferent biological functions (Dunn 2002; Simoes and Faro

2004; Timotijevic et al. 2010). In general, all non-viral APs

are synthesized as inactive precursors (zymogens), pre-

venting degradation during intracellular transport and

secretion (Dunn 1997). The removal of prosegment domain

during protease maturation process is an important feature

for enzyme activation. Besides this inhibitory function, the

prosegment is also involved in the proper folding and

intracellular sorting of the zymogen (Koelsch et al. 1994;

Dunn 1997; Horimoto et al. 2009). Like their animal and

microbial counterparts, plant AP sequences are encoded as

preproproteins with a signal peptide and a prosegment at

the N-terminus. However, typical plants APs display two

relevant differences in comparison with their animal and

microbial counterparts: (1) the consensus sequence of

catalytic triads and (2) the presence of an exclusive region

named plant specific insert (PSI). The catalytic triads of

typical plant APs are DTG/DSG instead of DTG/DTG

characteristic of those from animal and microbial origins,

except for BACE 1 and plasmepsin I and II (Francis et al.

1994; Vassar et al. 1999). The PSI is an insertion localized

at the C-terminal region of the protein and displays no

homology with other regions of APs from different origins.

This domain is partially or totally removed during the

enzyme maturation process, comprises approximately 100

aa and reveals high homology with saposins (mammalian

sphingolipids-activating proteins) (Glathe et al. 1998;

Ramalho-Santos et al. 1998; Duarte et al. 2008).

These similarities include six conserved cysteine residues,

a glycosylation site, similar hydrophobicity patterns and a

conserved tyrosine residue (Guruprasad et al. 1994;

Liepinsh et al. 1997). Although the physiological relevance

of the PSI was not yet established, its interaction with

membranes was demonstrated revealing an additional

ability of this domain in vesicles destabilization resulting in

vesicle leakage (Egas et al. 2000). The involvement in

membrane interactions and in the vacuolar sorting of APs

has been proposed by others (Tormakangas et al. 2001;

Terauchi et al. 2006). The complete sequencing of several

plant genomes revealed the existence of a large number of

new APs, with unique features. This identification led to a

reorganization of plant APs in typical, nucellin-like

and atypical proteinases. Actually, in MEROPS database

(http://merops.sanger.ac.uk), these newly identified plant

APs are grouped almost exclusively in sub-family A1B,

whereas typical plant APs are integrated in sub-family

A1A (Faro and Gal 2005; Rawlings et al. 2010). Although

typical APs have been extensively characterized from a

variety of plant species, there was no description of their

presence in green algae (Phylum chlorophyta). Given the

widespread nature and functional significance of these

enzymes in land plants it was thus considered of impor-

tance to demonstrate if typical APs are expressed in green

algae, and obtain some elucidation about the differences

between these proteases and their land plant homologues.

Chlamydomonas reinhardtii is a unicellular eukaryotic

green algae that is largely used as a model system in a

variety of molecular and cellular studies, mainly because of

its well-studied haploid genetic system and easy laboratory

manipulation (Harris 2009). This microalgae has been used

as an important tool in the study and elucidation of several

biological mechanisms such as photosynthesis, circadian

rhythms, chloroplast biogenesis and flagella assembly

(Grossman 2000; Mittag et al. 2005; Eberhard et al. 2008;

Wilson et al. 2008). In this work, we report the identifi-

cation and characterization of chlapsin, the only typical AP

encoded in C. reinhardtii genome. Although displaying

properties characteristic of typical plant APs, chlapsin

reveals unique features, like the unprecedented chloroplast

localization demonstrated by immunolocalization studies.

Differences between chlapsin and other typical plant APs

are discussed taking into consideration, this striking chlo-

roplast localization.

Materials and methods

Chlamydomonas reinhardtii library screening, chlapsin

cDNA isolation and sequence analysis

A commercial C. reinhardtii library constructed in the vector

Lambda Zap II was purchased from ‘‘Chlamydomonas

284 Planta (2012) 236:283–296

123

Genetic Centre’’ (http://www.chlamy.org) and used for

library screening. The cDNA was prepared from CC-1690

cells grown to middle log phase in HS medium bubbled with

5% CO2 and with an amplification titer of 2.2 9 109 pfu/ml.

The library was screened by plaque hybridization, using as a

probe an EST (AV632726), purchased from the ‘‘Kazusa

DNA Research Institute’’ (http://www.kazusa.jp). This EST

comprising the C-terminal and 30UTR region of a C. rein-

hardtii aspartic proteinase was 32P-labelled by random oli-

gonucleotide primed synthesis (Amersham MegaprimeTM

DNA labelling Systems, GE Healthcare). The library plaques

were transferred onto nylon filters (GE Healthcare) and

hybridized at 42�C for 48 h with the labelled probe. The

putative positive clones were isolated after two screening

rounds and their pBluescript phagemids were excised from

the Lambda Zap vector and the insert sequenced. The com-

plete sequence of the AP cDNA clone was obtained by

automated DNA sequencing of both strands. The nature of the

isolated cDNA was confirmed by using several bioinfor-

matics tools. Sequence similarity searches were determined

at UniProt (Jain et al. 2009; Consortium 2011) and multiple

sequence alignments were performed by using ClustalW

(Larkin et al. 2007; Goujon et al. 2010). The programme

SignalP (Emanuelsson et al. 2007) was used for signal peptide

prediction, the putative glycosylation sites were identified by

using the programme ScanProsite (Sigrist et al. 2010) and the

conserved domains were scanned at Pfam database (Finn

et al. 2010).

Recombinant expression, refolding and purification

of rchlapsin

The full-length cDNA of chlapsin was used as a template in

PCR amplification, in order to obtain a construct without

the putative signal peptide (chlapsinDpre). The primers

used in this amplification reaction were: 50-CGGCATA

TGGACCAGGGGCAGGTC-30 (forward primer) and

50-CCGCTCGAGCATAGCCGCATTGGC-30 (reverse pri-

mer), which included the restriction sites (underlined) for

NheI and XhoI, respectively. The chlapsinDpre construct

was subsequently subcloned into vector pET23a (Novagen)

and a positive chlapsinDpre_pET23a clone was confirmed

by DNA sequencing. This clone was then used as a tem-

plate to obtain a truncated form of chlapsin, without the

highly hydrophobic region comprising the predicted signal

peptide and the putative prosegment domain. This new

construct, chlapsinD(1–174 bp), hereby named rchlapsin,

was obtained by site-directed mutagenesis of chlapsinDpre_

pET23a, using the following primers: 50-AAGAAGGA

GATATACATATGGACCAGGGGCAGGTCACCCTA-30

(forward primer) and 50-TAGGGTGACCTGCCCCTGGTC

CATATGTATATCTCCTTCTTA-30 (reverse primer).

rChlapsin was transformed into BL21StarTM

(DE3)

Escherichia coli strain cells (Invitrogen), for protein

recombinant production. Expression, refolding, and purifi-

cation methods were adapted from Lin et al. (1993) and

Castanheira et al. (2005). Briefly, a recombinant clone

(rchlapsin) was cultured and allowed to grow at 37�C, to an

OD600 nm of 0.6. Recombinant rchlapsin was expressed

in the form of inclusion bodies, by addition of Isopropyl-

b-D-1-thiogalactopyranoside (IPTG) at a final concentration

of 0.5 mM. The cells were harvested by centrifugation 3 h

after induction, and the sediment was resuspended in 50 ml

of TN buffer (50 mM Tris–HCl pH 7.4, 50 mM NaCl)

supplemented with lysozyme (Sigma-Aldrich) at 100 lg/ml.

After a freezing/thawing cycle, the released DNA was

digested by addition of DNaseI (100 lg/ml) (Sigma-

Aldrich) and MgCl2 (100 mM) and the inclusion bodies

were washed with 1 l of TN buffer. A second washing step

was performed using TN buffer supplemented with 1%

Triton X-100. The final pellet was solubilized in 40 ml of

solubilization buffer (8 M urea; 0.1 M Tris–HCl; 1 mM

glycine; 1 mM EDTA; pH 10.5) containing 2-b-mercap-

toethanol (100 mM). The protein was diluted 209 in sol-

ubilization buffer supplemented with DTT (10 mM),

reduced glutathione (1 mM) and oxidized glutathione

(0.1 mM), and was refolded by dialysis for 48 h, against

three changes of 20 volumes of 20 mM Tris–HCl pH 8.0

buffer, at 4�C. The dialysed sample was concentrated in an

amicon ultrafiltration cell (Millipore) with a 10 kDa cut-off

membrane (YM-10) and the non-dissolved residues were

afterwards removed by ultracentrifugation at 40,000g for

20 min. The supernatant was applied onto an S-300 gel

filtration column (GE Healthcare), previously equilibrated

in 20 mM Tris–HCl pH 8.0 buffer supplemented with

0.4 M urea. The fractions corresponding to the non-aggre-

gated forms of rchlapsin were pooled and purified using an

ion-exchange chromatography, with a Mono-Q column

(GE-Healthcare). The recombinant protein was eluted

with a 0–0.5 M NaCl gradient, in buffer 20 mM Tris–HCl

pH 8.0 supplemented with 0.4 M urea, at a flow rate of

0.75 ml/min. Expression and purification of rchlapsin were

followed by SDS-PAGE, performed in a Bio-Rad ‘‘Mini

protean III’’ electrophoresis apparatus and using 12.5%

gels. The proteins were stained with 0.2% Coomassie

Brilliant Blue R 250 (GE-Healthcare).

Digestion of oxidized insulin b-chain

The enzymatic activity of purified recombinant rchlapsin

was tested towards oxidized insulin b-chain (Sigma-

Aldrich). Digestion of insulin was performed by incubating

purified rchlapsin and a partially purified fraction of native

chlapsin (unpublished results), with the oxidized insulin

b-chain (1 mg/ml), in 0.1 M formic acid pH 3 and 37�C.

The inhibitor pepstatin A at a final concentration of 1 lM

Planta (2012) 236:283–296 285

123

was added to the mixtures, in parallel experiments. The

reactions were stopped by the addition of trifluoroacetic

acid (TFA) at a final concentration of 0.6% (v/v) and then

centrifuged for 10 min at 12,000g. The resulting digestion

products were analyzed by reversed-phase high perfor-

mance liquid chromatography (RP-HPLC), in a C18

column (Kromasil 100 C18-Teknokroma), using a Promi-

nence HPLC system (Shimadzu LC-20AD). The digestion

products were separated by a linear gradient of 0–80%

acetonitrile in 0.1% TFA (v/v), for 35 min, at a flow rate of

1 ml/min and absorbance was monitored at 220 nm.

Enzymatic activity assays

The enzymatic activity of purified rchlapsin was also tested

towards five different fluorogenic peptides: typical AP

substrate (7-methoxycoumarin-4-acetic acid) (MCA)Lys-

Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys-(2,4-dinitrophenyl)

(DNP), BACE 1 substrate (MCA)Lys-Ser-Glu-Val-Asn-

Leu-Asp-Ala-Glu-Phe-Lys(DNP), renin substrate 1 Arg-

Glu(5-[(2 -aminoethyl)amino]naphthalene-1-sulfonic acid

(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(4-

(dimethylaminoazo)benzene-4-carboxylic acid (DABCY

L)-Arg (Sigma-Aldrich), HIV protease substrate 1 Arg-

Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DA

BCYL)-Arg (Sigma-Aldrich) and CDR1 protease substrate

(MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-

Lys(DPN). The peptide hydrolysis was monitored in a

spectrofluorimeter at an excitation wavelength of 328 nm

and an emission wavelength of 393 nm, for the peptides

with MCA/DNP. For those with EDANS/DABCYL, the

hydrolysis was monitored at an excitation wavelength of

335 nm and emission wavelength of 490 nm. To study the

pH influence on rchlapsin’s enzymatic activity, the assays

were performed against the CDR1 protease substrate using

the following buffers supplemented with 9% dimethyl

sulfoxide and 0.1 M NaCl: 50 mM sodium citrate buffer

pH 2.5, pH 3.0 and pH 3.5, 50 mM sodium acetate buffer

pH 4.0, pH 4.25, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5,

and 50 mM Tris–HCl pH 7.0. The effect of redox agents,

ions and nucleotide-related compounds on rchlapsin

enzymatic activity was assayed by pre-incubating the

enzyme with each compound, for 15 min, before determi-

nation of activity at pH 4.0, 37�C. Kinetics studies were

performed towards the same fluorogenic substrate-(MCA)

Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DPN)

at 37�C in buffer 50 mM sodium acetate pH 4.0 supple-

mented with 0.1 M NaCl and 9% DMSO. The active

enzyme concentration was determined by active site

titration, with the inhibitor pepstatin A (Verıssimo et al.

1996). The software Sigma-Plot was used for kinetic

parameters calculation based on the Lineweaver–Burk

plot.

Immunogold labelling

Chlamydomonas reinhardtii cells (approximately 50 ml of

cell suspension cultures) were centrifuged at 700 g for

3 min to pellet cells. Cells were resuspended in 1 ml of

fixative solution: 33 mM phosphate buffer pH 7.0, con-

taining 4% (v/v) paraformaldehyde and 0.6% (v/v) glu-

taraldehyde. Cells were washed in 33 mM phosphate

buffer pH7.0 and dehydrated in graded ethanol series from

50 to 90% (v/v) at 5 min intervals. After dehydration the

cells were incubated in graded series of LR-White resin

(medium grade-catalysed) and ethanol at room temperature

and during 25 min (mixtures 1:2, 1:1, 2:1, 1:0). The

embedded cells were polymerised at 55�C in LR-White,

using plastic bottleneck capsules. To pellet cells between

different solutions, the suspensions were centrifuged at

700g for 3 min. Sections for transmission electron

microscopy were obtained using a diamond knife and sil-

ver-coloured sections were placed in 400 mesh square

nickel grids. Ultrafine sections were incubated in TBS with

1% (v/v) tween 20 for 10 min followed by incubation in

TBS with 100 mM glycine. Grids were then placed in

blocking solution (TBS with 100 mM glycine and 0.25%

(v/v) goat serum, for 10 min and incubation with primary

antibodies (1:20 dilution in blocking solution) was per-

formed for 1 h, at room temperature. The primary antibody

used was designed against a polypeptide in the light chain

of the protein: CGGRVFPLRPEQYV. After rinsing in

blocking solution 59, grids were incubated for 1 h in gold

labelled goat anti-rabbit IgG (H ? L) (10 nm) diluted 209.

The final washing steps were performed in TBS (5 washes)

followed by 59 wash in distilled water. Sections were post-

stained in 2% (v/v) aqueous uranyl acetate saturated solu-

tion for 2 min and in lead citrate for 1 min. Observation

was carried out using JEOL JEM 1400 transmission elec-

tron microscope, operating at 60 kV.

Chloroplast isolation and fractioning

Intact chloroplasts were isolated based on the method

described by Mourioux and Douce (1981) with some

modifications. One litter of the algae culture was centri-

fuged at 700g for 3 min to pellet cells. Cells were resus-

pended in 120 ml of grinding buffer (0.33 M sorbitol,

0.05 M Na-Tricine, 10 mM MgCl2, 1 mM MnCl2, 10 mM

NaCl, 20 mM EDTA, 0.5 mM KH2PO4, 0.1% sodium

isoascorbate, final pH 8.0) and homogenised for 15 s.

Homogenate was filtered through miracloth and gauze

(4 layers each), centrifuged at 130g for 1 min to remove

the debris and then at 1,100g for 10 min to pellet chloro-

plasts. The pellet was resuspended in 4 ml of resuspension

buffer (330 mM sorbitol, 50 mM Hepes pH 7.6, 1 mM

MgCl2, 1 mM MnCl2, 2 mM NaNO3, 20 mM NaCl, 2 mM

286 Planta (2012) 236:283–296

123

EDTA, 5 mM sodium isoascorbate) using a soft brush.

Crude chloroplast fraction was washed three times with

50 ml of washing buffer (10 mM Na-Tricine, 10 mM

MgCl2, 10 mM NaCl, pH 8.0), resuspended in 20 ml of

digitonin buffer (10 mM Na-Tricine, pH 8.0, 10 mM NaCl,

10 mM MgCl2, 0.1 M sorbitol, 0.5% (w/v) digitonin), with

a soft brush, and incubated for 30 min on ice with stirring.

Insolubilized material was removed by brief centrifugation

(1,000g for 3 min) and then crude grana membranes were

pelleted (40,000g for 30 min). The supernatant contains the

stroma lamellae. Crude grana lamellae were resuspended

in 10 ml of washing buffer and 40 ml of Triton X-100. One

minute after mixing, the suspension was diluted 49 with

washing buffer and the undigested material removed

(1,000 g for 3 min). The supernatant fraction contains the

grana lamellae. Several samples were taken at different

time points of the procedure to work as controls in the

Western blot analysis: cells, culture media, cell debris,

crude chloroplasts, grana and stroma fractions.

Protein extraction and western blotting

Cell/chloroplast fractions were homogenized in 50 mM

sodium citrate pH 5.5, 5% (w/v) SDS, 0.01% (w/v) BSA,

150 mM NaCl and 2% (v/v) b-mercaptoethanol and boiled

for 10 min. Extracts were centrifuged at 12,000g for 30 min

at 6�C, and supernatants were collected. When necessary,

samples were TCA (trichloroacetic acid) precipitated: one

volume of sample was combined with one volume of 20%

(w/v) TCA. The mixture was homogenised in a vortex for

15 s and incubated on ice for 15 min. The mixture was

centrifuged at 12,000g for 15 min at 6�C and 500 ll of ice

cold acetone was added to the pellet, followed by a new

centrifugation step. The acetone step was repeated and

pellet was air dried. Protein fractions were combined with a

bromophenol based sample buffer, after protein quantifi-

cation (Bio-Rad protein assay). Samples containing about

15 lg of total protein were loaded onto 12.5% polyacryl-

amide gels for SDS-PAGE. Following electrophoretic

separation, proteins were blotted into a nitrocellulose

membrane using a tris-glycine-methanol buffer (25 mM

tris, 192 mM glycine, 20% methanol). For immunodetec-

tion the membranes were incubated in blocking solution

(5% (w/v) skim milk and 1% (w/v) BSA in Tris-buffered

saline (TBS), containing 0.6% (v/v) tween 20) for 1 h at

room temperature and then incubated overnight with a

1:1,000 dilution of the primary antibody (the same used in

immunogold labelling) in blocking solution, at 4�C. The

membranes were washed 39 in TBS, containing 0.1% (v/v)

tween 20, and incubated with 1:1,000 dilution of anti-rabbit

IgG conjugated to alkaline phosphatase (Vector) for

90 min, at room temperature. The membranes were washed

and detection was performed using a chromogenic substrate

for alkaline phosphatase-NBT/BCIP (Sigma). The purity

analysis of chloroplast and cytoplasmatic fractions was

performed using the protocol above mentioned with minor

alterations. The antibodies anti-NAB1 (Agrisera) and anti-

RbcL (Agrisera) were used at a dilution of 1:300, while the

anti-chlapsin antibody was used at a 1:500 dilution. The

secondary antibody, anti-rabbit (GE healthcare), was used

at a dilution of 1:10,000, and the detection was performed

using the chemiluminescent substrate for alkaline phos-

phatase-ECF (GE Healthcare).

Results

Isolation and sequence analysis of chlapsin cDNA

Isolation of full-length cDNA of chlapsin was performed

before C. reinhardtii genome sequence became available

and consequently required a cDNA library screening

approach. A commercial cDNA library prepared using

mRNA from C. reinhardtii was screened and one of the

positive clones revealed an AP nature, confirmed by com-

parison with other AP sequences. The nucleotide and

deduced amino acid sequences were deposited at EMBL/

Genebank database, with the accession number AJ579366.

Analysis of the nucleotide sequence revealed an open-

reading frame of 1,734 bp coding for a preproenzyme of

579 amino acids, hereby named chlapsin. Two untranslated

regions were also identified in the isolated cDNA sequence:

(1) a 50 UTR region of 147 pb, and (2) a 30 UTR of 566 pb

comprising a polyadenylation signal (TGTAA) which pre-

cedes the poly (A) tail. The ORF-derived aa sequence was

then subjected to sequence similarity searches using several

databases for identification of conserved domains. This

analysis revealed the presence of a putative signal sequence

of 23 aa, followed by a pro-region of about 42 aa inferred

from the alignment with other APs, two conserved Asp

residues D93 and D281 (PFAM:PF00026), two saposin-like

type B domains that mark the PSI region and two putative

glycosylation sites N129 and N470. This analysis revealed

that chlapsin shows the characteristic primary structure

organization of typical plant APs (Fig. 1).

Alignment of chlapsin with other plant APs

In general, APs are characterized by sharing a high

homology level and chlapsin is no exception revealing

higher identities with oryzasin-1 (accession number

Q42456) (40%) and phytepsin (P42210) (46%). Despite

this significant homology level, comparison of chlapsin’s

sequence with other plant APs revealed two major differ-

ences. One is the presence of identical catalytic triads

DTG/DTG, being this chlapsin feature shared with most

Planta (2012) 236:283–296 287

123

Fig. 1 Amino acid sequence

alignment of chlapsin and other

plant APs. Deduced chlapsin

amino acid sequence was

aligned with other plant APs

sequences using ClustalW

alignment programme. Both

catalytic triads are boxed and a

black bar underlines the PSI

region. The putative chlapsin

glycosylation sites are marked

with a circle. The regions

corresponding to the signal

peptide and prosegment are

highlighted by solid and dottedlines, respectively. Phytepsin

(accession number X56136)-

Hordeum vulgare; Oryzasin1

(accession number ACA50495)-

Oryza sativa; Cardosin A

(accession number AJ132884)-

Cynara cardunculus; PpAP1

(accession number AJ586914)

Physcomitrella patens and

Chlapsin (accession number

AJ579366)–Chlamydomonasreinhardtii

288 Planta (2012) 236:283–296

123

animal APs, as all typical plant APs display DTG/DSG

(Fig. 1) (Dunn 2002; Simoes and Faro 2004). The second

major difference between chlapsin and other APs resides in

the PSI region. Although chlapsin’s PSI comprises the

putative N-glycosylation site and the six conserved cyste-

ines typically found in these domains, this PSI domain is

180 aa long in contrast with the majority of plant PSIs

which have about 100 aa. This extra 80 aa sequence is a

unique segment, rich in alanine residues and localized

downstream the 3rd conserved cysteine residue (Fig. 1).

The PSI structure of phytepsin was already solved and

based on this structure, it is possible to infer that this new

segment of chlapsin’s PSI is localized in the loop region,

between the 3rd and 4th a-helices as shown in Fig. 2

(Kervinen et al. 1999). While maintaining the primary

structure organization of typical plant APs, chlapsin shows

some significant differences that clearly distinguish it from

other plant APs.

Expression and purification of recombinant chlapsin

(rchlapsin)

In order to characterize the enzymatic properties of

chlapsin, its cDNA without the putative signal peptide

(chlapsinDpre) was cloned into pET23a expression vector

for heterologous expression in E. coli. This first approach

was not successful as no significant protein accumulation

was observed in E. coli extracts (data not shown). In order

to circumvent this, a second approach was undertaken

involving the removal of the N-terminal segment Met1-

Ser60 that corresponds to a highly hydrophobic region,

comprising the predicted signal peptide and the putative

prosegment domain. rChlapsin was expressed in the form

of inclusion bodies and after a refolding process the

enzyme was purified by a size exclusion chromatography,

on a Sephacryl S300 column (Fig. 3a). This purification

step allowed the isolation of the non-aggregated forms of

rchlapsin (fraction IV) that were further purified by ion-

exchange with a MonoQ column (Fig. 3b). Fractions from

expression and purification steps were analysed by SDS-

PAGE (Fig. 3c) and eluted proteins were then tested for

enzymatic activity. The proteolytic activity of purified

rchlapsin was, initially, tested towards the peptide

(MCA)Lys-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP),

which is usually hydrolyzed by most typical APs from

several origins. Although sharing a high sequence simi-

larity with typical APs, purified rchlapsin was not able to

cleave that peptide, revealing a different enzymatic speci-

ficity. The identification of rchlapsin’s active fractions was

performed by an activity screening assay using several

fluorogenic peptides: BACE 1 substrate (MCA)Lys-

Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(DNP), renin

substrate 1 Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-

Val-Ile-His-Thr-Lys(dabcyl)-Arg, HIV protease substrate 1

Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys

(dabcyl)-Arg and CDR1 protease substrate (MCA)Lys-

Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DPN).

From this screening, rchlapsin was only able to cleave the

fluorogenic peptide designed for CDR1 protease (Simoes

et al. 2007) which allowed the identification of purified

rchlapsin active fractions. As shown in Fig. 3b, proteolytic

activity was mainly concentrated in fraction 2 that was

enriched in a protein with an apparent molecular mass of

50 kDa, as expected (Fig. 3c). rChlapsin was thus purified

in an active and pure form.

Enzymatic proteolytic activity of rchlapsin

In order to characterize rchlapsin’s proteolytic activity, the

effect of pH, temperature as well as the effect of several

protease inhibitors was studied using the CDR1 substrate

(MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-

Lys(DPN). The effect of pH on enzymatic activity was

tested by incubating purified rchlapsin at pH values from

2.5 to 7.0 (Fig. 4a). Recombinant rchlapsin exhibited a

maximum enzymatic activity at pH 4.25. A reduction in

almost 50% in activity was observed at pH 3.0 and pH 5.0.

At pH 3.5 the protease showed 60% of the optimal activity

and at pH 6.0 the protease retained only 10% of activity.

This pH profile resembles that of typical APs with higher

activity levels at more acidic pHs. The optimal tempera-

ture for the proteolytic activity of rchlapsin was also

Fig. 2 Comparison between the PSI domain of chlapsin and phytep-

sin. Alignment of PSI regions from phytepsin and chlapsin in order to

identify the localization of the 80 aa new insertion sequence in the PSI

structure of chlapsin. The 5 a-helices are represented by H1, H2, H3,

H4 and H5, respectively

Planta (2012) 236:283–296 289

123

determined (Fig. 4b). The hydrolysis of CDR1 substrate by

rchlapsin was significant for temperatures between 20 and

55�C, with maximum activity observed at 37�C. Above this

temperature, the activity gradually decreased and was

almost 10% at 70�C and for lower temperatures the activity

was residual below 20�C. The effect of specific inhibitors

characteristic of different classes of proteases on rchlapsin

activity was also evaluated. The proteolysis at acidic pH

was completely inhibited by the addition of pepstatin A,

Fig. 3 Purification of recombinant rchlapsin. a Size-exclusion chro-

matography of the refolded rchlapsin on a Sephacryl S-300 column.

The column was pre-equilibrated with 20 mM Tris–HCl pH 8.0

supplemented with 0.4 M urea (buffer A). Elution was performed at a

0.5 ml/min flow rate and the fractions were detected at 280 nm.

b Ion-exchange chromatography of fraction IV, corresponding to non-

aggregated rchlapsin. The fraction was applied onto a Mono-Q

column and eluted using a linear gradient of 0–0.5 M NaCl, in buffer

A. The enzymatic activity was tested towards the fluorogenic peptide

(MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-Lys(DPN)

and is represented by a discontinuous line. c SDS-PAGE analysis of

the expression and purification process, stained with Coomassie

Brilliant Blue. Lane 1 total protein extract before induction, lane 2,

total protein extract after induction, lane 3 inclusion bodies, lane 4fraction IV from size exclusion chromatography, lane 5, fraction 2

from Mono Q

Fig. 4 Effects of pH, temperature, and protease inhibitors on

recombinant rchlapsin enzymatic activity. a The effect of pH was

assayed by incubating the enzyme at 37�C with buffers from pH 2.5 to

pH 7.0 (50 mM sodium citrate pH 2.5 to pH 3.5; 50 mM sodium acetate

buffer pH 4.0 to pH 6.5; 50 mM Tris–HCl pH 7.0) supplemented with

0.1 M NaCl and 9% dimethyl sulfoxide. b The temperature effect on

recombinant rchlapsin activity was tested by incubating the enzyme at

different temperatures for 15 min. c The effect of protease inhibitors on

rchlapsin activity was tested by preincubating the enzyme with each

inhibitor for 15 min, at 37�C and pH 4.0. In all cases, the enzymatic

activity was tested towards the fluorogenic peptide (MCA)Lys-Leu-His-

Pro-Glu-Val-Leu-Glu-Lys(DPN)

290 Planta (2012) 236:283–296

123

thereby reinforcing the nature of chlapsin as a typical AP.

No significant inactivation occurred when the enzyme was

incubated with EDTA, E-64 or Pefabloc (Fig. 4c). The

CDR1 substrate was also used for the determination of

kinetic parameters. The Km and kcat values, at pH 4.0, were

estimated to be 0.75 lM and 0.35 s-1, respectively. The

Km was very similar to that described for CDR1, support-

ing the use of this peptide for rchlapsin enzymatic char-

acterization (Simoes et al. 2007). In order to further

characterize rchlapsin’s specificity, oxidized insulin

b-chain was also used as a substrate. The insulin digestion

resulted in only two major peptides that were resolved by

RP-HPLC, using a C-18 column (Fig. 5). Each peptide was

analysed by mass spectrometry and a single cleavage site

was identified (*Leu15–Tyr16*). Insulin cleavage by

rchlapsin was restricted to one site, further corroborating

the lack of proteolytic activity over 4 of the 5 synthetic

substrates tested. This strict specificity towards oxidized

insulin b-chain was further confirmed by making use of a

partially purified fraction of native chlapsin (data not

shown). As shown in Fig. 5, a similar profile was obtained

and enzymatic activities of both native and recombinant

chlapsin were completely inhibited in the presence of

pepstatin A, as expected (Fig. 5). rChlapsin exhibited

properties typical of the AP class, as a maximum enzy-

matic activity at acidic pH and a complete inhibition by the

archetypical inhibitor pepstatin A. However, the restricted

specificity observed for rchlapsin clearly differentiate this

enzyme from typical plant APs which in general display

broader specificity requirements, anticipating a more spe-

cialized role for this new enzyme.

Chlapsin localization

Chlapsin subcellular localization on C. reinhardtii cells

was achieved using two approaches, one complementing

the other. In a first stage, immunogold labelling was per-

formed revealing clear chloroplast localization (Fig. 6a).

The labelling was detected all over the Chlamydomonas

chloroplast (Fig. 6b) and no labelling was detected either in

the cytoplasm or other organelles. To confirm these results

and get a finer localization, chloroplast isolation was car-

ried out and samples taken during the procedure were used

for Western blotting. Results showed no chlapsin accu-

mulation in the growth media or in cell debris or other

organelles than chloroplast (Fig. 7, lanes M and Deb). Two

bands with approximately 25 and 30 kDa, corresponding

probably to intermediate forms of the maturation process of

the enzyme, were detected in cells and chloroplast fractions

(Fig. 6c, lanes C and Chl). To evaluate whether chlapsin is

located in chloroplast stroma or bond to chloroplast

membranes, crude chloroplasts extracts were fractionated

and stroma and grana fractions were analysed separately.

Chlapsin was only detected in the stroma fraction (Fig. 7a,

lane Str), being not detected in grana lamellae (Fig. 7a,

lane Lam). An immunoblot using compartment markers

Fig. 5 Enzymatic activity of

both native and recombinant

chlapsin towards oxidized

insulin b-chain. a Purified

rchlapsin was incubated with

oxidized insulin b-chain, the

digestion products were

separated by RP-HPLC and the

preferred rchlapsin cleavage

sites were identified by mass

spectrometry. b A partially

purified fraction of native

chlapsin was also incubated and

digestion products separated by

RP-HPLC for comparison

purposes. Two parallel assays

were performed in the presence

of the inhibitor pepstatin A

(discontinuous line). The

sequence of oxidized insulin

b-chain and the identified

cleavage sites are shown in the

bottom. 1 and 2 correspond to

digestion fragments analyzed by

MS and 3 to non-cleaved

oxidized insulin b-chain

Planta (2012) 236:283–296 291

123

was performed in order to confirm the nature and purity of

each fraction and reinforce the chloroplast localization of

chlapsin (Fig. 7b).

Additives effect on rchlapsin enzymatic activity

The activities of chloroplast proteases are largely regulated

by the presence of redox agents, ions, and nucleotides

(Sakamoto 2006; Balsera et al. 2010). Based on this, and

given the unexpected chloroplast localization of chlapsin,

the proteolytic activity of recombinant rchlapsin was tested

in the presence of the referred additives (Table 1). Of all

cations studied, Fe2? showed a strong inhibitory effect of

about 50%. All the other mono, di and trivalent cations had

no effect on rchlapsin activity. Moreover, rchlapsin was

significantly inhibited by the presence of NADH and

almost completely inhibited by the presence of NADPH.

The oxidized counterparts NAD and NADP displayed no

effect on proteolytic activity. The enzymatic activity of

rchlapsin was also affected by the presence of nucleotides

dATP and dGTP and purine type deoxynucleotides.

Discussion

Plant APs have been detected in different organs and tis-

sues in a large number of land plants and most of them

showed a vacuolar or extracellular localization (Simoes

and Faro 2004). Despite the considerable amount of

knowledge on family A1A plant homologues, there is still a

lack of information regarding the presence of these

Fig. 6 Chlapsin localization in

Chlamydomonas reinhardtiicells. a Immunogold labelling

of chlapsin using anti-chlapsin

specific antibody shows a

markedly chloroplast

localization. Bar corresponds to

1 lm. b Detail of C. reinhardtiichloroplast evidencing chlapsin

labelling. C chloroplast;

M mitochondria, N nucleus

Fig. 7 Immunoblot of Chlamydomonas chloroplasts isolation and

fractioning. a Immunoblot analysis, using anti-chlapsin specific

antibody, of crude cells (C),culture media (M), cell debris (Deb),

crude chloroplasts (Chl), grana lamellae (Lam) and stroma (Str).

Chlapsin was detected in the intact cells and stroma fractions but not

in the culture media or in the grana lamellae. b Western-blot analysis

of the efficiency of fractioning process. The antibodies anti-NAB1

(cytoplasm marker), anti-RbcL (chloroplast marker) were used as

compartment markers (upper panel) and anti-chlapsin antibody was

used to confirm chlapsin localization (lower panel)

292 Planta (2012) 236:283–296

123

important enzymes in the green algae group. In this work,

we report the identification and characterization of the first

typical AP from the green algae, C. reinhardtii. This

new AP, named chlapsin (accession number AJ579366)

revealed an interesting and unexpected chloroplast locali-

zation. This is the first time a typical AP is identified in this

organelle which turns chlapsin unique among plant typical

APs. Chlapsin displays several other distinctive features in

comparison with typical plant APs, raising new questions

regarding evolution and functional specialization of this

vast group of plant enzymes.

In terms of primary structure, chlapsin shares several

common characteristics with other typical plant APs but

with unique striking features. The catalytic motif Xaa-Xaa-

Asp-Ser/Thr-Gly-Xaa, where the Xaa is a hydrophobic

amino acid which is present in both chlapsin catalytic triads

but instead of the conserved DTG/DSG motifs found in all

typical plant APs, chlapsin harbors the pair DTG/DTG more

common in their animal and microbial counterparts.

In chlapsin, the PSI domain is composed by six cysteine

residues and a putative glycosylation site, like almost PSIs

described so far. However, chlapsin’s PSI is significantly

longer. The PSI domain of chlapsin is 180 aa long, and has a

unique insertion of about 80 aa, rich in alanine triplets,

between the 3rd and 4th a-helix, corresponding to the PSI

loop region. This insertion will probably not alter the PSI

structure since this loop region is a disordered region that is

projected away from the helical bundle (Kervinen et al.

1999). Interestingly, the newly released genome sequences

of a few Chlorophyta organisms like Volvox carteri,

Chlorella variabilis, and Micromonas pusilla disclosed the

presence of at least one AP encoding gene with DTG/DTG

triads and a PSI with 130–170 aa, about 30 to 70 aa longer

than the typical ones (Worden et al. 2009; Blanc et al. 2010;

Prochnik et al. 2010). Apart from this extra segment, these

PSI domains maintain in their primary structure the six

conserved cysteine residues, the putative glycosylation site

and the tyrosine residue present in the linker between the

first and the second a-helices. The functional significance of

these longer PSI domains is currently unknown but it

appears to be characteristic of APs in the Phylum chloro-

phyta, bringing new features to this family of proteases.

Most APs are synthesized as inactive zymogens and the

maturation process requires the removal of the N-terminal

region, named prosegment (Dunn 2002). In this work,

recombinant chlapsin was expressed without the first 60 aa

zymogen sequence which would correspond to the putative

signal peptide and prosegment domain (rchlapsin). Our

results clearly demonstrate that the absence of chlapsin’s

prosegment was not detrimental for its folding and activity.

This emerges as another distinctive feature of chlapsin

since this domain was shown to be required for the proper

folding of the large majority of typical APs. The enzymatic

activity of rchlapsin was studied revealing a maximum

activity at pH 4.25 and an optimal temperature of 37�C.

Additionally, pepstatin A completely inhibited rchlapsin

enzymatic activity as expected. Inhibitors from other

classes of proteases showed no inhibitory effect, reinforc-

ing the nature of rchlapsin as a typical AP. Despite these

characteristics, rchlapsin displayed a narrow enzymatic

specificity in comparison with other typical plant APs.

From all substrates tested, rchlapsin cleaved only the

substrate specifically designed for CDR1 and oxidized

insulin b-chain at the bond Leu15–Tyr16. The cleavage of

this peptide bond was also described for several other plant

APs, but was always combined with additional hydrolysis

sites (Kervinen et al. 1993; Verıssimo et al. 1995; Mazorra-

Manzano et al. 2010). This restricted specificity was con-

firmed when a partially purified fraction of native chlapsin

was tested and a similar digestion profile was observed.

This restricted activity towards insulin is shared with

Arabidopsis CDR1 and Wheat GIAP2, and suggests a

Table 1 Effect of different compounds on the rchlapsin enzymatic

activity

Compound Concentration

(mM)

Enzymatic

activity (%)

Standard

deviation

Redox agents Control 97.3 2.6

DTT 1 94.1 9.8

GSH 2 115.5 10.8

GSSG 2 117.1 11.9

NAD 3 94.2 2.4

NADH 3 5.1 1.5

NADP 2 87.9 2.6

NADPH 2 4.1 2.0

Nucleotides ATP 1 70.4 5.8

ADP 1 84.6 5.3

GTP 1 52.4 4.7

CTP 1 75.6 7.5

dATP 1 67.0 3.4

dGTP 1 62.9 2.9

dCTP 1 91.0 2.9

dTTP 1 84.6 1.7

Ions NaCI 20 87.8 4.6

FeCI2 2 40.1 9.5

CaCI2 2 106.4 1.2

MnCI2 1 99.7 6.6

MgCI2 2 104.3 3.8

ZnAc2 1 100.8 4.9

MnCI3 2 97.5 12.0

Purified rchlapsin was pre-incubated for 15 minutes with each com-

pound, at pH 4.0, 378C. The enzymatic activity was assayed towards

the fluorogenic peptide (MCA)Lys-Leu-His-Pro-Glu-Val-Leu-Glu-

Lys(DPN) and the activity of the control sample was taken as 100%.

Values are means of three replications with standard error

Planta (2012) 236:283–296 293

123

tightly regulated enzymatic activity. This is particularly

relevant considering chlapsin’s chloroplast localization and

the specific and sequential proteolytic events involved in

chloroplast metabolism (Bleukx and Delcour 1999;

Sakamoto 2006; Simoes et al. 2007; Eberhard et al. 2008).

Indeed, the localization of chlapsin in Chlamydomonas

chloroplast raises very interesting questions and brings the

hypothesis of chlapsin participation in chloroplast mainte-

nance or degradation events. Although some intra-plastid-

ial proteolytic functions have been described like: signal

peptide removal, amino acid acids recycling, removal of

damaged proteins like D1 from photosystem II, and puta-

tive role in regulation of plastid gene expression, this is the

first time a typical AP is detected in chloroplast (Kato et al.

2004, 2005; Hortensteiner 2006; Sakamoto 2006; Eberhard

et al. 2008; Olinares et al. 2011; Tanaka and Tanaka 2011).

The detection of native chlapsin revealed the presence of

probable intermediate forms of enzyme’s maturation pro-

cess. The conversion of zymogenes into smaller forms

through sequential cleavage steps is a common mechanism

of activation of typical APs (Dunn 2002; Castanheira et al.

2005). The chloroplast metabolism depends largely on

redox agents and nucleotides, and proteases involved in

that metabolism are usually regulated by these compounds

(Sakamoto 2006; Balsera et al. 2010; Malyan 2010). In a

similar way, rchlapsin proteolytic activity was also sus-

ceptible to the presence of NADH, NADPH, dATP, dGTP,

ATP and GTP. The redox agents, in their reduced form,

inhibited almost completely rchlapsin activity and the

purine nucleotides reduced proteolytic activity in about

30%. Interference of NADH and ATP on enzymatic

activity of rchlapsin suggests that this enzyme may have a

putative housekeeping function contributing to the

dynamic of photosynthetic function.

According to the primary structure of chlapsin, a strong

signal peptide is predicted at the N-terminus suggesting that

this enzyme might be targeted to the chloroplast through a

recently identified pathway involving ER-Golgi-plastid

protein trafficking (Villarejo et al. 2005; Radhamony and

Theg 2006). However, a dual targeting to ER and chloro-

plast as described to RB60, cannot be excluded for chlapsin

(Levitan et al. 2005). In fact, a potential dual localization of

chlapsin could help explaining the marked difference in the

number of APs encoding genes in C. reinhardtii when

compared with higher plants. Only three AP genes are

present in the Chlamydomonas genome whereas a large

representation of APs genes was found in land plants; as 73

APs homologues in Arabidopsis thaliana, about 96 homo-

logues in Oryza sativa, 26 AP genes in Physcomitrella

pattens genome, and 100 homologues in Vitis vinifera

genome (Faro and Gal 2005; Chen et al. 2009; Rawlings

et al. 2010). Although C. reinhardtii is a unicellular

organism in contrast with land plants that possess different

tissues and organs, the difference in APs gene number is

excessively marked (D’Hondt 1993; Runeberg-Roos et al.

1994; Asakura et al. 1995; Vieira et al. 2001; Chen et al.

2002). This striking low number of APs genes in C. rein-

hardtii constitutes an intriguing issue and suggests multiple

uses of translated APs, probably accumulating different

functions and/or localizations. Chlamydomonas simplicity

as a unicellular organism will likely help to clarify in the

future several of the herein raised questions, like chlapsin

sorting pathway to the chloroplast or its putative role in the

organelle’s protease network.

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