The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

Abstract

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

Full text

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R E S E A R C H L E T T E R
Thechaotrope-solubleglycoproteinGP1isaconstituentofthe
insolubleglycoproteinframeworkoftheChlamydomonas cellwall
J¨ urgen Voigt1,2, Ronald Frank3& Johannes W¨ ostemeyer2
1Institute for Biochemistry, Charit´ e, Paul Ehrlich Centre for Experimental Medicine, Berlin, Germany;2Institute for Microbiology, Friedrich-Schiller-
University, Jena, Germany; and3Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Correspondence: J¨ urgen Voigt, Institute for
Biochemistry, Charit´ e, Paul Ehrlich Centre for
Experimental Medicine, Monbijoustrasse 2,
D-10117 Berlin, Germany. Tel.: 149 30 450
528 144; fax: 149 30 450 528 942; e-mail:
juergen.voigt@charite.de
Received 8 September 2008; accepted 17
November 2008.
First published online 19 December 2008.
DOI:10.1111/j.1574-6968.2008.01456.x
Editor: Claire Remacle
Keywords
antibodies; Chlamydomonas reinhardtii; cell
wall; epitope mapping; glycoprotein; insoluble
wall fraction.
Abstract
Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell
wall component, a sac-like framework of cross-linked glycoproteins containing
22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only
polypeptide with an even higher proportion of hydroxyproline (35%) occurring in
vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released
from the purified insoluble cell wall fraction by trypsin treatment and epitope
analyses of polyclonal antibodies raised against different deglycosylation products
of this particular wall fraction using 181 chemically synthesized GP1-derived
pentadecapeptides revealed evidence that GP1 is indeed a constituent of the
insoluble wall component.
Introduction
The cell wall of the unicellular green alga Chlamydomonas
reinhardtii does not contain cellulose or other polysacchar-
ides, but consists exclusively of an insoluble, hydroxypro-
line-rich glycoprotein (HRGP) framework and several
chaotrope-soluble, hydroxyproline-containing
teins (Roberts, 1974; Imam et al., 1985). A multilayered
architecture was observed for the walls of C. reinhardtii as
revealed by electron microscopical studies (Roberts et al.,
1972, 1985; Roberts, 1974; Goodenough & Heuser, 1985).
In addition to an ‘inner’ wall of covalently cross-linked
HRGPs reminiscent of the higher plant hydroxyproline-rich
meshworks (Roberts et al., 1985), they possess an ‘outer
wall’ of HRGPs that coassemble into crystalline arrays. The
arrays can be solubilized by treatment with aqueous solu-
tions of chaotropic salts, and they reassemble when the
chaotropic salts are removed by dialysis (Hills et al., 1975;
Goodenough & Heuser, 1985; Goodenough et al., 1986;
Adair et al., 1987). These HRGPs consist of c. 20 different
polypeptides (Imam et al., 1985) and can be extracted from
intact C. reinhardtii cells by treatment with aqueous solu-
glycopro-
tions of chaotropic salts like LiCl (Voigt, 1985a, 1988). One
of the major constituents of this particular cell wall fraction
is the chaotrope-soluble glycoprotein GP1 (Ferris et al.,
2001). GP1 is a monomeric glycoprotein with a molecular
mass of 272359Da and a carbohydrate content of 76%
(Ferris et al., 2001). Its polypeptide backbone (65129Da)
(Ferris et al., 2001) contains 32.3% hydroxyproline as
revealed by amino acid analysis of the purified mature
glycoprotein (Goodenough et al., 1986). This value even
exceeds the proportion of hydroxyproline in the insoluble
cell wall fraction (Vogeler et al., 1990). The amino acid
sequence of GP1 derived from the nucleotide sequence of
the gp1 gene (Ferris et al., 2001) consists of four domains.
The (hydroxy)proline-rich sequences are located in domain
2, which contains 49 repeats of the PPSPX motif, and
domain 3, which consists of PS repeats (Ferris et al., 2001).
Both domains form the 100-nm-long fibrous structure of
this molecule (Goodenough et al., 1986; Ferris et al., 2001),
whereas the globular head (Goodenough et al., 1986) can be
attributed to the C-terminal domain (domain 4).
As recently reported, the chaotrope-soluble, HRGP GP2
is a precursor of the insoluble glycoprotein frameworkof the
FEMS Microbiol Lett 291 (2009) 209–215
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C. reinhardtii cell wall (Voigt et al., 2007). Therefore, we have
investigated whether or not GP1 is an additional constituent
of the insoluble cell wall fraction of C. reinhardtii.
Materials and methods
Strains and growth conditions
The C. reinhardtii Dangeard wild-type strain 137C (mating
type1) was obtained from Dr R. P. Levine (Harvard
University, Cambridge, MA). Cells were grown at 211C and
20000 lux in a high-salt medium supplemented with 0.2%
(w/v) sodium acetate as described previously (Voigt &
M¨ unzner, 1987). Cells were harvested by centrifugation at
6000g for 10min at 41C and washed twice with fresh
culture medium.
Isolation of the chaotrope-soluble cell wall
glycoproteins
The chaotrope-soluble cellwall glycoproteins GP1, GP2, and
GP3 were extracted from C. reinhardtii wild-type cells and
separated as previously described (Voigt, 1985a,b; Good-
enough et al., 1986; Voigt et al., 1996, 2007).
Purification of the insoluble cell wall component
The insoluble cell wall component of C. reinhardtii was
isolated from intact wild-type cells by treatment with
different detergent-containing buffers essentially as recently
described (Voigt et al., 2007).
Chemical deglycosylation
Deglycosylation of the freeze-dried insoluble cell wall com-
ponent was performed by treatment with trimethylsilyl
trifluoromethane sulfonate in anhydrous trifluoroacetic acid
containing thioanisol or anhydrous hydrogen fluoride (HF)/
pyridine as previously described (Vogeler et al., 1990; Voigt
et al., 2007).
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
Macromolecules released from the insoluble cell wall fraction
were precipitated by addition of trichloroacetic acid (final
concentration 10%, w/v) in the cold and the precipitates
collected by centrifugation. The pellets were washed twice with
distilled water and redissolved in a small volume of urea–SDS
buffer (Voigt, 1985a,b). After addition of sample buffer
(Laemmli, 1970) containing bromophenol blue as tracking
dye, the polypeptides were separated by SDS-PAGE according
to Laemmli (1970) on gel slabs (83mm?65mm?0.15mm)
containing 12% (w/v) acrylamide. After electrophoresis, the
gels were stained with Coomassie brilliant blue G250.
Epitope analysis
A total of 181 overlapping pentadecapeptides representing
the whole amino acid sequence of the Chlamydomonas GP1
protein derived from the ORF of the GP1 cDNA (GenBank
accession no. AF309494) were generated by spot synthesis
using a cellulose paper sheet as solid support (Frank, 1992).
These overlapping pentadecapeptides were used to deter-
mine the epitope specificities of the different antibodies as
previously described (Voigt & Frank, 2003; Voigt et al.,
2007). After incubation with the antibody and extensive
washing, bound rabbit immunoglobulin G (IgG) were
measured by incubation with alkaline phosphatase-coupled
goat anti-(rabbit IgG) immunoglobulin (Amersham-Buchler,
Braunschweig, Germany) and subsequent detection of the
indirectly bound alkaline phosphatase via its enzyme activity
using 5-bromo-4-chloro-3-indolyl phosphate as substrate in
the presence of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT). After documentation of the
results, the membrane was stripped as previously described
(Frank, 1992).
MS
Proteolysis with trypsin of the purified insoluble cell-wall
fraction was performed essentially as described for in-gel
digestion of protein bands cut from SDS-PAGE gels (Shev-
chenko et al., 1996; Kratzer et al., 2008). The cell-wall
material was digested for 3h with porcine trypsin (sequen-
cing grade, modified; Promega, Madison, WI) at a concen-
tration of 67ngmL?1in 25mM ammonium bicarbonate, pH
8.1, at 371C. Before peptide mass mapping and sequencing
of tryptic fragments by tandem MS, peptide mixtures were
extracted from the insoluble cell-wall material by 1% formic
acid followed by two changes of 50% acetonitrile. The
combined extracts were vacuum-dried until only 1–2mL
were left and desalted using ZipTips according to the manu-
facturers’ instructions (Millipore, Bedford, MA). Matrix-
assisted laser desorption ionization reflection time of flight
(MALDI-ReTOF) analysis from the matrix a-cyano-4-
hydroxycinnamic acid/nitrocellulose prepared on the target
using the fast evaporation method (Arnott et al., 1998;
Kratzer et al., 2008) was performed on a Bruker Reflex IV
(Bruker Daltonik, Bremen, Germany) equipped with a N2
337-nm laser and gridless pulsed ion extraction. Sequence
verifications of the fragments were performed by nanoelec-
trospray tandem MS on a Q-TOF I mass spectrometer
(Micromass, Manchester, UK) equipped with a nanoflow
electrospray ionization source. Gold-coated glass capillary
nanoflow needles were obtained from Protana (Type Med-
ium NanoES spray capillaries, Odense, Denmark). Database
searches (NCBInr, nonredundant protein database) were
performed using the MASCOT software from Matrix Science
(Perkins et al., 1999).
FEMS Microbiol Lett 291 (2009) 209–215
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210
J. Voigt et al.

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Amino acid analyses
Amino acid analyses wereperformed as previouslydescribed
(Vogeler et al., 1990).
Results
Polypeptides of the insoluble cell wall
component
The insoluble cell wall component of C. reinhardtii isolated
from intact cells by successive treatments with different
detergent-containing buffers showed a sac-like morphology
as revealed by phase-contrast microscopy (Fig. 1a). This
morphology was completely destroyed by chemical deglyco-
sylation with HF/pyridine accompanied by solubilization of
distinct polypeptides (Fig. 1b). This polypeptide pattern was
highly reproducible but dependent on the cell cycle stage
(Fig. 1b, lanes 1 and 2).
Edman degradation of the constituents of the insoluble
cell wall fraction released by chemical deglycosylation with
either HF/pyridine or trimethylsilyl trifluoromethane sulfo-
naterevealed thattheir N-terminal amino acid sequences are
rather heterogeneous. Mixtures of several amino acid resi-
dues are cleaved off during each round of Edman degrada-
tion (Voigt & Frank, 2003; Voigt et al., 2007). These findings
are at least partly due to the fact that the polypeptide
constituents of the insoluble wall fraction are cross-linked
not only via the HF-sensitive bonds but also via HF-resistant
isodityrosine formation and transglutaminase-dependent
reactions (Waffenschmidt et al., 1993, 1999).
The chaotrope-soluble hydroxyproline-rich cell
wall glycoprotein GP1 is a constituent of the
insoluble cell wall component
As previously reported, polyclonal antibodies raised against
deglycosylation products of the insoluble cell wall fraction
preferentially react with the chaotrope-soluble cell wall
glycoproteins GP2 and GP3B (Voigt et al., 2007). Further-
more, GP2 was found to be a precursor of the insoluble
glycoprotein framework of the Chlamydomonas cell wall
(Voigt et al., 2007). Question arised whether or not the
chaotrope-soluble, hydroxyproline-rich cell wall glycopro-
tein GP1 is another constituent of the hydroxyproline-rich
insoluble wall fraction. To answer this question, we started
with an immunochemical approach. To this end, 181 over-
lapping pentadecapeptides, together representing the entire
polypeptide backbone of GP1, were synthesized on a solid
support (Fig. 2a and b) and comparatively analyzed for
binding of polyclonal antibodies raised against the chemi-
cally deglycosylated, chaotrope-soluble and -insoluble cell
wall fractions (Fig. 2c). The polyclonal antibody raised
against the chemically deglycosylated, chaotrope-soluble cell
wall glycoproteins (anti-dCSCW) reacted with almost all
pentadecapeptides derived from domains 1 (peptide nos
1–11) and 4 (peptide nos 124–181) but not with those
derived from the proline-rich domains 2 and 3 (Fig. 2c,
upper panel). This holds true also for the antibodies raised
against the deglycosylation products of the insoluble cell
wall component (Fig. 2c, lower panels). However, the anti-
dICW antibody additionally recognized the pentadecapep-
tides 83–87 and 89–93 derived from sequences of the
proline-rich domain 2 of GP1. Anti-dICW-64kDa and
anti-dICW-45kDa reacted with other pentadecapeptides
derived from sequences of the proline-rich domain 2 of
GP1 (peptide nos 29–30 and 53–56) in addition to those
derived from domains 1 and 4 (Fig. 2c, lower panels). The
conclusion that at least the C-terminal domain 4 of GP1 is
contained in the insoluble glycoprotein framework of the
C. reinhardtii cell wall was further corroborated by mass
spectrometrical analyses of peptides released from the highly
purified insoluble cell wall component by trypsin treatment
(Table 1). MALDI-ReTOF analyses of these tryptic peptide
Fig. 1. The insoluble glycoprotein framework of the Chlamydomonas
reinhardtii cell wall. (a) Phase-contrast micrograph of the insoluble cell
wall component of C. reinhardtii. Scale bar=10mm. (b) SDS-PAGE
analysis of polypeptides released from the purified insoluble cell wall
component of C. reinhardtii by treatment with anhydrous HF/pyridine.
Cultures of the C. reinhardtii wild-type strain 137C (1) were synchro-
nized by growth for 3–4 days under a constant light-dark regime (14h
light–10h darkness). Half of the cells were harvested at the end of the
cell division phase (about 6h after beginning of the last dark period)
when the daughter cells were still surrounded by the mother cell wall
(=sporangia). The other half of the cultures were transferred to the light
and harvested as soon as all the daughter cells (=zoospores) were
released from the sporangia by degradation of the sporangia walls
(=cell walls of the mother cells). The insoluble cell wall fractions were
prepared from both cell cycle stages and subjected to chemical deglyco-
sylation as described in the Materials and methods section. Deglycosyla-
tion products of the insoluble wall fraction corresponding to 30mg of
protein were fractionated by SDS-PAGE on slab gels containing 12%
acrylamide and subsequently stained with Coomassie brilliant blue
G250. Lane 1, insoluble wall fraction isolated from sporangia; lane 2,
insoluble wall fraction isolated from zoospores.
FEMS Microbiol Lett 291 (2009) 209–215
c ?2008 Federation of European Microbiological Societies
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211
The insoluble wall fraction of Chlamydomonas contains GP1

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Fig. 2.
FEMS Microbiol Lett 291 (2009) 209–215
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212
J. Voigt et al.

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fragments revealed the presence of 12 peptides that are
derived from the C-terminal domain of GP1 and cover
73% of this particular GP1 sequence (Table 1). The amino
acid sequences of these tryptic peptides were verified by MS/
MS data obtained by nanoelectrospray tandem MS (data not
shown). Because several pentadecapeptides derived from the
N-terminal domain of GP1 are recognized by our polyclonal
antibodies raised against the deglycosylation products of the
insoluble cell wall component (Fig. 2c), we assume that the
whole GP1 is a constituent of the insoluble wall component.
The high proportion of hydroxyproline (c. 35%) in GP1 is
presumably the reason for the high hydroxyproline content
(c. 22%) of the insoluble wall component (Table 2), which is
considerably higher than that of GP2 (14.7%), which was
recently found to be a constituent of the insoluble glycopro-
tein framework of the C. reinhardtii wall (Voigt et al., 2007).
Discussion
As shown in this study, at least the C-terminal domain 4 of
the chaotrope-soluble cell wall glycoprotein GP1 is a con-
stituent of the insoluble glycoprotein framework of the
C. reinhardtii cell wall. Most of the chemically synthesized
pentadecapeptides derived from domain 4 of GP1 were
recognized by polyclonal antibodies raised against deglyco-
sylation products of the insoluble cell wall component
(Fig. 2c, lower panels). Furthermore, MALDI-ReTOF ana-
lyses of peptides released from the highly purified insoluble
wall component by trypsin treatment revealed the presence
of 12 fragments, which together cover 73% of the C-terminal
domain of GP1 (Table 1). The amino acid sequences of these
particular peptides were verified by mass spectrometric
sequencing (data not shown).
The polyclonal antibody raised against the deglycosy-
lated, chaotrope-soluble cell wall polypeptides not only
recognized the chemically synthesized pentadecapeptides
covering the C-terminal domain but also those derived from
the N-terminal domain (Fig. 2c, upper panel). This finding
was astonishing in so far as the predominant proportion of
this domain was assumed to be the leader peptides of GP1
(Ferris et al., 2001), which should be cleaved off cotransla-
tionally. Several pentadecapeptides derived from domain 1
were also recognized by the polyclonal antibodies raised
against deglycosylation products of the insoluble cell wall
component indicating that domain 1 of GP1 is also con-
tained in the insoluble glycoprotein framework of the
Chlamydomonas cell wall. Therefore, we assume that the
(hydroxyl)proline-rich domains 2 and 3 are also present in
the insoluble wall component. Most of the proline-rich
synthetic pentadecapeptides derived from domains 2 and 3
(peptides 13–124) were not recognized by the polyclonal
antibodies raised against the deglycosylation products of the
chaotrope-soluble and insoluble wall components (Fig. 2c).
This finding was not astonishing because proline residues
located in proline-rich motifs are usually substrates for the
proline hydroxylases (Tanaka et al., 1981; Blankenstein et al.,
1986; Kieliszewski & Lamport, 1994). Indeed, amino acid
analyses of GP1 revealed a proportion of c. 35% hydroxy-
proline (Table 2), which is considerably higher than the
proportion of hydroxyproline found in the insoluble wall
component (c. 22%). For comparison, GP2, the recently
described precursor of the insoluble wall fraction (Voigt
et al., 2007), only contains c. 15% hydroxyproline (Table 2).
These findings indicate that GP1 is the precursor of the
insoluble wall component that causes its rather high
Table 1. GP1-derived peptide fragments detected by MALDI-TOF ana-
lyses in the tryptic digests of the purified insoluble cell wall fraction of
Chlamydomonas reinhardtii
Sequence?
Positionw
[M1H]1
D
ObservedCalculated
K.LVWADDAIAFDDLN-
GTSTRPGSASR.M
R.MVGEPDIAGTK.C
K.GNLKGWMPKPSR.N
K.GNLKGWMPKPSRN-
PR.W
K.GWMPKPSR.N
K.GWMPKPSRNPR.W
R.WGQAVFSGGR.T
R.TVGSVANVTIR.V
R.VAFATEKPALIYSSIEL-
VVYNTGATLIR.V
R.VPIAANVTR.S
R.INMGAGNK.K
K.TSIDAVGLNLK
386–4102636.88662636.83770.0498
411–421
424–435
424–438
1118.3917
1371.7423
1739.1375
1118.2963
1371.6495
1739.0577
0.0954
0.0928
0.0798
428–435
428–438
439–448
449–459
460–487
959.2568
1326.6533
1065.2549
1117.3902
3041.6131
959.1602
1326.5684
1065.1766
1117.2907
3041.5578
0.0966
0.0849
0.0783
0.0995
0.0553
488–496
534–541
545–555
941.2081
805.0376
1131.4146
941.1187
804.9457
1131.3145
0.0894
0.0919
0.1001
?The amino acid residues located upstream and downstream, respec-
tively, from the detected peptides in the GP1 sequence (GenBank
accession number S22477) are separated by points.
wPosition in the amino acid sequence of GP1 (reference Ferris et al., 2001).
Fig. 2. Comparative epitope analyses of the polyclonal antibodies raised against the deglycosylated, chaotrope-soluble cell wall fraction (anti-dCSCW)
and different polypeptides of the insoluble cell wall fraction (anti-dICW, anti-dICW-64kDa, and anti-dICW-45kDa). (a) Schematic representation of the
deduced amino acid sequence of GP1. The (hydroxyl)proline-rich domain is indicated by a black box, the (Ser-Pro)n-rich domain by a gray box. Numbers
indicate the positions of pentadecapeptides and amino acid residues from the N-terminus. (b) Overlapping pentadecapeptides (181) derived from the
ORF of the GP1 cDNA (GenBankTM/EBI accession number AF309494) and representing the whole known amino acid sequence of GP1 were synthesized
by spot synthesis using cellulose paper as a solid support (Frank, 1992). (c) After treatment with blocking solution, the cellulose-bound scan peptides
were incubated with the different antibodies. After extensive washing, bound IgGs were detected by incubation with alkaline phosphatase-coupled
anti-rabbit IgG antibodies and subsequently visualization of the indirectly bound alkaline phosphatase as described under Materials and methods.
FEMS Microbiol Lett 291 (2009) 209–215
c ?2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
213
The insoluble wall fraction of Chlamydomonas contains GP1

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proportion of hydroxyproline. However, not all proline
residues in the proline-rich domains 2 and 3 of GP1 are
hydroxylated. Some proline-rich synthetic pentadecapep-
tides reacted with the anti-dICW antibody (peptides 83–87
and 89–93) and some others with anti-dICW-64kDa and
anti-dICW-45kDa, respectively (peptides 29–30 and 53–
56). The nonhydroxylated proline residues apparently occur
in the motifs SPAPPSPA (position 90–97), PPVPPSP (posi-
tion 165–170), PPSPKP (position 256–261), and RPPFPA
(position 277–282).
The molecular massof
(65129Da) (Ferris et al., 2001) is rather similar to the
64-kDa component released from the insoluble wall fraction
by chemical deglycosylation (Fig. 1b). Amino acid analyses
of the 64-kDa and 45-kDa constituents of the insoluble wall
fraction, however, revealed that they contained o6%
hydroxyproline (data not shown). Therefore, both polypep-
tides cannot be truncated forms of the GP1 backbone
(c. 35% hydroxyproline), but must contain several frag-
ments of the GP1 backbone. This conclusion is corroborated
by the findings that polyclonal antibodies raised against
both constituents of the deglycosylated insoluble wall frac-
tion (anti-dICW-64kDa and anti-dICW-45kDa, respec-
tively) cross-react with GP2 and GP3B (Voigt et al., 2007).
thedeglycosylated GP1
Furthermore, both polypeptides cross-react with a polyclo-
nal antibody raised against a C. reinhardtii 14-3-3 protein
(Voigt & Frank, 2003). Edman degradation of both poly-
peptide components revealed that they are not individual
peptides because several amino acid residues were cleaved
off during each round of Edman degradation (Voigt &
Frank, 2003). Taken together, these findings indicate that
both polypeptide constituents of the insoluble wall fraction
are rather heterogeneous and consist of fragments of differ-
ent polypeptide precursors (including GP1) which are
presumably cross-linked via peroxidase-catalyzed intermo-
lecular isodityrosine formation (Waffenschmidt et al., 1993)
and/or transglutaminase-dependent reactions (Waffensch-
midt et al., 1999). Both cross-linking types are stable under
the conditions of chemical deglycosylation. It has been
reported that partial proteolysis of the insoluble glycoprotein
framework of the Chlamydomonas cell wall is a prerequisite
for cell enlargement and is accompanied by release of macro-
molecules into the culture medium (Voigt, 1985b, 1986). The
size of the polypeptide fragments retained in the insoluble
cell-wall layer is apparently not only determined by the
distribution of putative cleavage sites but also by the carbo-
hydrate chains and the associated chaotrope-soluble cell-wall
glycoproteins (Voigt & Frank, 2003; Voigt et al., 2007).
Table 2. Amino acid composition of GP1 and other cell wall constituents
Amino acid
GP1
GP2GP3
Insoluble wall
component FoundCalculated?
Mol%w
Number Mol%?
Number?
Mol%w
Mol%w
Mol%w
Hyp (O)
Pro (P)
Ala (A)
Cys (C)
Asp (D)
Glu (E)
Phe (F)
Gly (G)
His (H)
Ile (I)
Lys (K)
Leu (L)
Met (M)
Asn (N)z
Gln (Q)z
Arg (R)
Ser (S)
Thr (T)
Val (V)
Trp (W)
Tyr (Y)
34.77?1.52
3.78?0.32
11.53?0.44
0.68?0.03
4.14?0.21
1.44?0.07
1.62?0.07
4.81?0.28
0.32?0.03
3.03?0.14
2.68?0.21
2.70?0.14
1.14?0.09
–
–
2.50?0.33
15.20?0.39
3.50?0.15
5.20?0.31
ND
0.86?0.19
193
21
64
37.48
2.52
11.17
0.72
1.26
0.54
1.26
4.14
0.18
2.70
2.34
2.34
1.26
2.52
0.72
2.34
15.68
3.78
5.41
0.72
0.72
208
14
62
14.73?0.59
7.74?0.23
8.54?0.29
1.31?0.08
8.08?0.34
7.28?0.35
3.82?0.17
8.52?0.39
0.23?0.02
1.80?0.07
3.03?0.22
7.04?0.29
1.04?0.07
–
–
3.02?0.12
8.26?0.36
6.59?0.27
5.69?0.23
ND
3.58?0.11
5.74?0.23
4.54?0.19
10.24?0.51
4.89?0.37
10.56?0.47
7.43?0.31
3.60?0.14
10.96?0.53
0.96?0.09
3.19?0.15
4.46?0.21
6.97?0.32
0.85?0.05
–
–
2.08?0.03
9.33?0.44
7.37?0.29
5.85?0.24
ND
1.18?0.02
21.87?0.94
5.07?0.21
8.15?0.33
1.09?0.08
7.46?0.32
6.16?0.27
3.28?0.11
7.65?0.33
1.09?0.12
2.58?0.15
3.88?0.17
6.36?0.29
0.99?0.07
–
–
2.49?0.04
8.95?0.39
5.86?0.22
4.87?0.16
ND
2.19?0.05
3.8
23
8
9
26.7
1.8
16.8
14.9
15.0
6.3
4
7
3
7
23
1
15
13
13
7
–
–
14
4
13.9
84.4
19.4
28.9
ND
4.8
13
87
21
30
4
4
?Calculated on the basis of the assumption that all proline residues in the proline-rich domains are glycosylated.
wGiven values are means?SD of six experiments.
zAsn and Gln are hydrolysed to Asp and Glu, respectively, during treatment with HCl.
ND, not determined.
FEMS Microbiol Lett 291 (2009) 209–215
c ?2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
214
J. Voigt et al.

Page 7

Acknowledgement
We are grateful to Ulrich Kratzer and many biochemical
students of the University of Tuebingen for their support to
obtain the mass spectroscopical data.
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The insoluble wall fraction of Chlamydomonas contains GP1