Eur. J . Biochem.

170,485-491 (1987)
0 FEBS 1987
Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii
are (hydroxy)proline-specific proteases
Lothar J AENICKE, Wolfgang KUHNE, Rainer SPESSERT, Ursula WAHLE and Sabine WAFFENSCHMIDT
Institut fur Biochemie, Universitat zuKoln
(Received J une 12iAugust 28, 1987) - EJ B 87 0686
Two stage specific cell-wall lytic enzymes (autolysins) from different strains of the unicellular, biflagellated
green alga Chlamydomonas reinhardtii were isolated and purified to homogeneity. Quantitative and specific
photometric assays for biological activity were worked out to follow fractionation and to establish lytic specificity
and kinetics. The autolysins were studied for enzymatic properties and screened for biological activity towards
several wall components obtained by salt extractions of sporangia and zoospores from C. reinhardtii. The
autolysins are proteolytic enzymes, fragmenting proline- or hydroxyproline-containing polypeptides in structures
like connective tissue. They attack predominantly selected domains within the walls of zoosporangia or gametes.
The sporangial autolysins are not only site- and strain-specific but also stage-specific, whereas the gamete
autolysins lyse cell walls of gametes as well as those of sporangia and zoospores.
Lysis of cell walls is an essential event in the course of
life of higher and lower plants [l]. It is the precondition for
germination, growth, differentiation, fertilization and conju-
gation. In the vegetative and sexual cycles of the unicellular
flagellated chlorophyte Chlamydomonas reinhardtii there are
three stages in which cell wall lysis by the action of lytic
enzymes (autolysins) occurs: (a) a sporangial autolysin re-
leases the zoospores from the maternal sporangium; (b) a
gamete autolysin dissolves the cell wall of the gamete prior to
fusion; (c) an as-yet-unidentified autolysin opens the
zygospores when the products of meiosis hatch. The
autolysins involved in the first two processes seem to be extra-
ordinarily specific. According to Schlosser [2], they are (a)
strain-specific, i.e. they discriminate between cells of different
strains of the genus Chlamydomonas; (b) stage-specific, i.e.
they recognize the developmental stage of the algae. The
sporangial autolysin dissolves only the wall of sporangia; the
gamete autolysin acts on the walls of gametes, zoospores and
sporangia as well.
The gamete autolysin is secreted during agglutination of
mating-type plus (mt') and minus (mt-) gametes. I t is par-
ticularly interesting, because it produces protoplasts from
zoospores [3]. It has been studied by several groups [4 - 71 and
finally described by Matsuda et al. [8] as a 62-kDa zinc-
requiring protease. Crude extracts of the sporangial autolysin
are obtained [2] by exposing suspensions of synchronized
sporangia (1 O8 cells/ml) to white light which releases the
zoospores. After spinning down, the cell-free supernatant has
sporangial lytic activity in a calcium-dependent protease of
37 - 40 kDa [ 9] . Thus, stage-specific wall autolysins are pro-
teases of defined but different molecular properties. Compari-
Correspondence to L. J aenicke, lnstitut fur Biochemie der
Universitat zu Koln, An der Bottmuhle 2, D-5000 Koln 1, Federal
Republic of Germany
Abbreviation: RBB, Remazol brilliant blue.
Enzymes. a-Galactosidase (EC 3.2.1.22); P-galactosidase (EC
3.2.1.23); galactose oxidase (EC 1.1.3.9).
son of the two autolytic systems at the enzyme and the sub-
strate level should provide a better insight into the mechanism
of wall lysis, may explain the strain and stage specificity and
should help to unravel the complicated composition of algal
cell walls.
We describe here the isolation and characterization of the
sporangial autolysins from two cross-reacting strains of C.
reinhardtii and the gamete autolysin of the same splitting
group [2]. To follow purification and for enzyme kinetic stud-
ies, we have developed quantitative methods for the
sporangial and gamete autolysin based on the liberation of
wall fragments and protoplasts, respectively. Crude extracts
and the purified autolysins were assayed for their hydrolytic
activity towards synthetic substrates in order to characterize
enzymatic properties and towards fractions of cell wall prep-
arations of isotypic and allotypic C. reinhardtii in order to
explore their sites of attack and their splitting requirements.
MATERIALS AND METHODS
Materials and instruments
All reagents and enzymes were purest grade obtainable
from Boehringer-Biochemica (Mannheim, FRG), Merck
(Darmstadt, FRG), Serva (Heidelberg, FRG) or Sigma
(Taufkirchen, FRG) ; adsorbents were products of Bio-Rad
Laboratories (Miinchen, FRG) for Biogel P 200, Whatman
(Hormuth-Vetter, Wiesloch, FRG) for DEAE-cellulose (DE-
52) and Miles (Bayer Diagnostic, Miinchen, FRG) for hydro-
phobic column-filling material. Spectrophotometric measure-
ments were made with a UV-120-02 from Shimadzu
(Diisseldorf, FRG), fluorescence determinations with a
Fluorispec from Baird Atomic (The Hague, Holland, NL).
Electrophoreses were run in the vertical electrophoresis unit
2001, from LKB-Producter (Stockholm, Sweden). Sonication
was performed with the Labsonic 1510 from Braun
(Melsungen, FRG).
486
Growth of cultures
Vegetative cultures. Cells of strain 13 32 aM and 77 81 of
the Algensammlung Gottingen were provided by Dr Schlosser
and grown in a 20 mM Tris/acetate/phosphate-containing me-
dium pH 7.6, with Hutners mineral solution as described in
[lo]. The algae were synchronized at 26C by a fixed cycle of
12-h dark and 12-h light (10 klx) and by daily dilution to lo6
cells/ml (11 32 aM) or 0.5 x lo6 cells/ml (77 81) after hatch-
ing. Aerated cultures were grown in 10 ml (test tubes), 300 ml
or 1000 ml medium. Cultures synchronized under these con-
ditions form sporangia in the dark: strain 11 32 aM after 2 h;
strain 77 81 after 4 to 5 h.
Sexual cultures. Strains 137 c (mt') and 137 c (mt-) were
obtained from Y. Matsuda and grown as described for veg-
etative cultures. Gametes were induced on agar plates (accord-
ing to a personal communication from Y. Matsuda) using
medium of reduced (20%) nitrogen concentration.
t
Preparation of autolysins
Preparation of crude sporangial autolysin. Sporangia were
centrifuged (500 x g, 20 rnin), resuspended to 100-fold initial
count, and zoospores forced to release by stopping aeration
and starting light again. The process was controlled under
the microscope. The hatched zoospores were sedimented at
10000 x g for 15 min. The supernatant (crude extract) can be
stored in 0.1% NaN3 at 4C without loss of activity for at
least one week.
Preparation of crude gamete autolysin. Equal amounts of
gametes of each mating type (4 x l o7 cells/ml) were mixed and
allowed to mate for 30 min. Cell-free crude gamete autolysin
was obtained by centrifugation at 12000 x g for 20 min. After
lyophilization extracts can be stored for weeks without loss
of activity.
Assay of cell-wall lytic activity
Activity assays of the sporangial autolysin were performed
in 0.02 M Tris/HCl buffer pH 8.2 with 0.1 mM Ca2+ and
1 mM Mg2' (Tris/Ca2'/Mg2+buffer) of gamete autolysin in
0.02 M borate buffer pH 9.0 with 0.1 mM Ca2' and 1 mM
Mg2 + .
Biological assays. Synchronized sporangia were killed by
adding 0.025% formaldehyde, washed twice with the above
Tris/Ca2'/MgZ' buffer pH 8.2 and resuspended to lo6 cells/
ml. 0.1 ml of this suspension was incubated for 20 rnin at
37C with 0.1 ml of the sample. After stopping lysis by the
addition of 0.01 ml 0.2 M EDTA, released zoospores were
counted and compared with a sporangia blank in buffer alone.
Quantitative assay f or the sporangia autolysin. Cells were
stained with Remazol brilliant blue (RBB) in a modification
of the staining procedure for xylans [12] as described for
Chlorella by Touet and Aach [13]. To a stirred suspension of
4 x lo9 sporangia and 0.01 g RBB in 30 ml water, 10 ml25%
sodium sulfate was added within 10 min; then 10 ml 25%
NaOH was poured and stirring continued for another 20 min.
The dark-blue sporangia were washed with Tris/Ca2 '/Mg2 +
buffer pH 8.2 and suspended to a concentration of l o7 cells/
ml. For use 1 ml of the suspension was centrifuged, decanted
and resuspended in 0.04 ml of the same buffer. 0.2 ml sample
was added, incubated at 37 "C and stopped after 20 rnin with
0.2 ml 0.2 M EDTA. After centrifuging at 13000 x g, the
supernatant was measured at 605 nm against a blank without
enzyme. The spectra of a standard incubation before and after
+ A [am1
400 500 600 700
Fig. 1. Remazol brilliant blue assay for sporangia autolysin. lo7 RBB-
stained sporangia were incubated with 0.2 ml sporangial autolysin as
described in Materials and Methods. Non-lysed cells were centrifuged
and the supernatant (curve a) measured against a blank with incu-
bation buffer (curve c) . The spectrum of the dye (10 pM in incubation
buffer) is shown in curve b
lysis, and of the free dye are shown in Fig. 1. One sporangial
autolysin unit is defined as the amount of enzyme that pro-
duces an increase of absorbance at 605 nm of 0.002/20 min.
Quantitative assay ,for the gamete autolysin. Wemodified
the procedure of Snell [6] which is based on the photometric
determination of chlorophyll released from protoplasts after
the addition of the detergent Triton X-100. Synchronized
zoospores of strain 137 c (mt') (10- 12 h after hatching) were
killed by formaldehyde as described above, washed with
0.02 M borate/Ca2'/Mg2+buffer pH 9 and resuspended in
the same buffer at 4 x l o7 cells/ml; 0.3 ml of this suspension
was incubated with 0.2 ml sample for 10 min at 37C. The
reaction was stopped by addition of 0.5 ml 0.1% Triton X-
100 in incubation buffer containing 10 mM EDTA. After
10 min at 4 T , non-lysed cells were removed at 13 000 x g and
the supernatant measured at 440 nm against a blank without
enzyme. One gamete autolysin unit is defined as the amount
of enzyme producing an increase of absorbance at 440 nm of
O. l / l O rnin.
Preparation of cell walls
Preparation of zoospore walls. The walls are stripped off
the cells by sonication at 100 W under cooling in an ice-bath
at I-min intervals for at least 5 min. Cell walls were sedimented
at 15000 x g for 20 min and washed twice with distilled water.
Preparation of sporangia walls. The wall is shed off the
sporangia by sonication at 50 W for 5 rnin in 1-min intervals.
Each time it is controlled microscopically that only sporangia
are opened and zoospores left intact. Zoospores were then
removed in a bench-top centrifuge at 700 x g for 5 min. The
turbid supernatant was pipetted off and centrifuged at
12000 x g for 10 min. The sporangial wall fragments were
washed twice with distilled water. Wall components were ex-
tracted overnight from the pellet with 10 vol. 2 M sodium
perchlorate according to Catt et al. [15]. The perchlorate-
soluble portion was fractionated by column chromatography
on Sepharose 2B and Sepharose 6B with 2 M sodium per-
chlorate as the eluant [I 61. The perchlorate-insoluble fraction
(fraction A) was then extracted at 70C for 2 h with the above
volume 0.3% sodium chlorite in 0.12% acetic acid by bubbling
with Nz [27, 281. By centrifugation for 10 min at 12000 x g
the soluble portion (fraction B) is separated from the insoluble
pellet (fraction C).
Galactosidase-treated walls. 1000 ml of synchronous spo-
rangia (lo6 cells/ml) were killed by the addition of
487
formaldehyde (see above), centrifuged (500 x g/20 min),
washed twice with water and incubated with 0.2U of the
appropriate enzyme for 10 h at 37C in 5 ml 0.02 M Tris/
0.01 mM Ca2+/1 M Mg2+ buffer. After incubation, sporan-
gia were centrifuged again and washed several times and used
for biological tests as described before.
Purification methods
Ion-exchange chromatography. A column (25 cm x 2 cm)
of DEAE-cellulose (DE-52) equilibrated with 0.02 M am-
monium acetate buffer pH 5.0 was loaded with 20 mg protein
in up to 10 ml buffer. Bound protein was eluted with a linear
gradient of 0 - 1 M NaCl in 0.02 M ammonium acetate buffer
pH 5.8; 2-ml fractions were collected and active fractions were
pooled.
Hydrophobic column chromatography. C-4-aminoalkyl-
Sepharose (2 ml) was used, equilibrated with 0.05 M borate
buffer pH 8.2 (1 mM Mg2+ and 0.1 mM Ca"). Adsorbed
protein was eluted by a linear 0 - 1 M NaCl gradient in the
same buffer.
Ge1,filtration. A Biogel P-200 column (50 cm x 2 cm) was
equilibrated with 100 mM borate buffer pH 8.5 containing
1 mM Mg2+ and 0.1 mM Ca2+, charged with the protein and
the eluent fractionated in 3-ml portions. Active fractions were
pooled.
Protein determination
Protein was assayed by the modified method of Lowry et
al. [29] with bovine serum albumin as the standard.
Determination f or carbohydrates with bicinchoninate
Determination of reducing sugars was done with the modi-
fied 2,2'-bicinchoninate method [23] with D-glucose as the
standard.
Other enzyme assays
Endoglycosidases. 0.5 ml enzyme preparation and 0.5 ml
polysaccharide solution (2%, xylan or mannan [24]) in 0.02 M
Tris buffer pH 8.2 (0.1 mM Ca2+, 1 mM Mg") were incubat-
ed for 60 inin at 37C. 0.5 ml of the incubated samples were
assayed by the bicinchoninate method [23] to measure release
of reducing sugars against blanks with heat-inactivated en-
zyme.
Exoglycosidases [24]. Substrates were the commercial
(Sigma, Taufkirchen, FRG) o-nitrophenyl derivatives of WD-
and P-D-galactose, a-L-arabinose, P-D-xylose and fl-D-glUCOSe.
Protrolytic activity. We followed the standard fluor-
escamine procedure described by Allen [25] ; calibration was
done with a standard of L-leucine (0-200 nmol/ml), or with
azocoll as the substrate as described by Walter [26].
Electrophoretic methods
For denaturing SDSjPAGE we used the discontinous sys-
tem of Laemmli [30], with a 6.5% stacking gel and 12%
resolving gel (10-cm length). Gels were run at 35 mA (stacking
gel) and 40 mA (resolving gel), then stained for protein with
0.1 % Coomassie brilliant blue R 250 in acetic acid/methanol/
water (10:45:45, v/v/v) and developed in acetic acid/
methanol/water (10: 20: 70, v/v/v).
RESULTS
Quantitative assay methods
Reliable quantitative and labour-saving assays for the
sporangial and gamete autolysins are required to follow the
purification procedure and kinetics economically. The tedious
methods reported to date [2,4,7,14] count released zoospores
or protoplasts after incubation with a given amount of en-
zyme. To save time and enzyme, we worked out photometric
procedures for both activities.
Sporangia autolysin. The dye Remazol brilliant blue (R BB)
is used to stain polysaccharides as substrates for endo-
glycosidase assay [12, 171. Touet and Aach introduced this
method to analyze lytic activities in Chlorella [13]. The modifi-
cation described in Materials and Methods allows this pro-
cedure to be applied to wall-stained Chlamydomonas spo-
rangia. During incubation of RBB-stained sporangia with
sporangial autolysin preparations, wall fragments are released
as indicated by the characteristic absorption maximum of the
dye at 605 nm, E =9.25 x l o3 M-' cm-', which was used for
quantification. The amount of released fragments is linear in
a wide range with time and enzyme concentration. Substrate
saturation was at lo7 sporangia/assay ; optimal incubation
temperature was 37"C, and a 20-min incubation time gave
sufficient increase in absorbance.
Gamete autolysin. The gamete autolysin does not discrimi-
nate between the walls of gametes, zoospores and sporangia.
Thus any of them might be used as substrate. Accordingly,
in previous studies on this activity, either sporangia were
incubated with gamete autolysin, and released zoospores were
counted [IS - 201 or protoplasts resulting after incubation of
zoospores for a given time were lysed with 0.1 Yn Triton X-100
[4, 21, 221or with 1.5 M sorbitol[22] or by boiling [7] and the
surviving cells counted. Snell 161modified the procedure of
Claes by measuring the chlorophyll released after Triton lysis.
Weadapted this method to our system and found 0.05% of
the tenside optimal and a wavelength of 440 nm for photom-
etry most convenient, and thus derived the protocol described
in Materials and Methods. The test is linear with enzyme
concentration and time and can be used to measure activity
at enzyme concentrations between 0 - 10 pg/sample.
Purification of autolysins
The purification steps and yields are listed in Table 1. They
are detailed in Materials and Methods.
Sporangial autolysins. Acetone was added from 20% to
50% to 400ml crude extract of synchronous sporangia of
strain 77 81 of specific activity 1900 U/mg protein. This
yielded 31.5 mg protein with a specific activity of 3200 U/
mg protein. On ion-exchange chromatography on DEAE-
cellulose the activity is displaced at 0.075 M NaC1. This results
in a purification factor of 16. Subsequent hydrophobic
chromatography on C,-aminoalkyl-Sepharose at a gradient
concentration of 0.1 M NaCl yielded 0.7 mg of electro-
phoretically pure protein as a symmetrical peak. This purified
sporangial autolysin had a specific activity of 52 140 U/mg
protein, corresponding to 27.4-fold purification. A similar
protocol (see Table 1) starting with 200 ml crude extract of
synchronous sporangia of strain 11 32 aM with a specific
activity 1570 U/mg protein using ammonium sulfate precipi-
tation between 30% and 65%, ion-exchange chromatography
on DEAE-cellulose (elution between 0.6 M and 0.8 M NaCl)
and gel-exclusion chromatography on Bio-Gel P-100 gave the
sporangia autolysin 12.7-fold purified at a constant specific
488
Fig. 2. SDS gel electrophoresis of purified sporangial autolysin from strain 77 81 (lanes a and b) and of gamete autolysin strain 137 c (lanes c
and d) . Lane a, active fractions from hydrophobic column (0.01 mg protein); lane b, after DEAE-cellulose (0.025 mg protein); lane c, active
fractions from DEAE-cellulose (0.03 rng protein); lane d, Bio-Gel P-200 (0.02 mg protein). Numbers indicate molecular mass in Da
Table 1. Purification of sporangial and gamete autolysins of C. reinhardtii strains
Autolysin Step Volume Protein Activity Yield Activity Factor
specific
Sporangial
from
77 81
Sporangial
from
11 32aM
Gamete
from
137 c
ml mg kU % U/mg -fold
crude extract 400 53.2 100 100 19 900 1 .o
acetone-preci-
pitation (20- 500/,) 50 31.5 95 95 3 020 1.6
DEAE-cellulose
(0.075 M NaCl) 10 2.0 61 61 30 380 16.0
hydrophobic
column (0.1 M NaCI) 5 0.7 37 37 52140 27.4
crude extract 200 21 33 100 1570 1
ammonium sulfate
(30-65%) 50 4.9 30 90 5 959 3.8
DEAE-cellulose
(0.06 -0.08 M NaCI) 20 1 .o 12.5 40 12 000 7.6
Bio-Gel P-100
(fr. 17-20) 6 0.5 10.6 30 20 000 12.7
crude extract 200 176 40 100 2 300 1
ammonium sulfate
(30-7OYo) 200 44 40 100 9 100 4
DEAE-cellulose
(0.1 M NaCI) 200 8 30 75 38000 16.7
Bio-Gel P-200
(fr. 17-20) 12 1.2 9 33 75 000 33
activity of 2000 U/mg. The protein is electrophoretically uni-
form (Fig. 2a, b). Its heat-inactivation kinetics is first order,
indicating one active species in the enzyme preparation.
Gamet e autolysin. 200 ml crude supernatants of mixed mt +
and mt- gametes of strain 137c with 176mg protein and
a specific activity of 2300 U/mg were enriched fourfold by
ammonium sulfate precipitation between 30% and 70% satu-
ration. After dialysis the resulting protein (44 mg) was bound
to DEAE-cellulose ion-exchanger and the autolysin activity
eluted with 0.1 M NaCl. Chromatography on Bio-Gel P200
( Vo =46 ml) gave 1.2 mg electrophoretically homogenous
protein (Fig. 2c, d) 33-fold enriched. The glycoprotein nature
of gamete autolysin was demonstrated by treatment with
trifluoromethanesulfonic acid, by which reducing sugars were
liberated (arabinose, galactose, glucose, mannose).
Characteristics of autolysins
Size-exclusion chromatography data and SDS-electro-
phoretical analysis showed that both the sporangial and the
gamete autolysins are single-chain molecules, the former with
a relative molecular mass of 39 - 40 kDa, the latter of 67 kDa.
They are stable at 37C. At 50C and 30-min incubation in
the respective incubation buffers, the sporangial autolysin has
489
70..
60-
50-
LO.-
30..
20.-
10..
*I.
activity
l : q 80
A
PH
Fig. 3. pH optima ofChlamydomonas autolysins. pH optima of (A)
gamete autolysin of strain 137 c and (B) sporangia autolysin of strain
1 1 32 aM were measured following the standard procedures described
in Materials and Methods, but using incubation buffers with pH
between 3 and 11. Up to pH 7 we used 0.02 M phosphate buffer
containing 0.1 mM Ca2+ and 1 mM Mgz+. A control was performed
to show that phosphate did not interfere with the activity of autolysins
lost 32% of its initial activity and the gamete autolysin is
totally inactive. The inactivation follows in both cases first-
order kinetics. Both lysing enzymes have their pH optimum
at 37C at slightly alkaline range as detailed in Fig. 3, the
sporangial autolysins having a narrow one around pH 8.2
(Fig. 3B), the gamete autolysin a broad one around pH 9.0
(Fig. 3A). They are stable at the tested pH for several hours.
Both enzymes show rather similar general behaviour
towards inhibitors and activators, the gamete autolysin
always being more sensitive than the sporangia autolysin
(Table 2). They are inhibited by EDTA; the sporangial
autolysins needing higher concentrations (1 mM) for total
block. Inhibition can be partly overcome by the addition
of CaZ+ and Mg2+. The autolysins are inhibited by
o-phenanthroline. The heavy metal chelator 2,2-dipyridyl is
only effective at extremely high concentrations. Phos-
phoramidon, an endo-metalloprotease inhibitor found by
Matsuda [8] to be a potent inhibitor of the gamete autolysin,
has only slight action on the gamete autolysin but acts more
strongly on the sporangial autolysins. This leaves doubts to
the heavy-metal requirement of these enzymes. SH reagents
also have no effect at usual concentrations (up to 2mM).
Serine-specific modifiers, such as phenylmethylsulfonyl fluor-
ide at millimolar concentration, interfere with lysis; again
the gamete autolysin is the more sensitive. General protease
inhibitors like a,-macroglobulin and aprotinin decrease the
activity of both types of autolysins to some extent, whereas
some specific enteric protease inhibitors such as leupeptin,
pepstatin, chymostatin and antipain have no effect. The ga-
mete autolysin, however, is strongly inhibited by the chymo-
trypsin-specific inhibitor tosylphenylalanine chloromethylke-
tone. Inhibitors, which are typical for collagenases, and also
Table 2. Effect of inhibitors on cell-wall autolysins of Chlamydomonas
reinhardtii
All data are mean values of five separate samples, accuracy of
measurement was +5%. n.d. =not determined
Inhibitor Concn Inhibition of autolysin
sporangial gamete
strain
strain strain 137 c
11 32 7781
mM %
EDTA 0.4 55 50 100
+Ca2 + 55 80 35
o-Phenanthroline 0.2 70 70 100
2,2-Dipyridyl 2.0 45 n.d. 35
Phenylmethylsulfonyl fluoride 2 75 60 100
Tosylphenylalanine
chloromethylketone 2 75 n.d. 100
Tosylly sine
chloromethylketone 2 35 n.d. 50
Lectin from: mg/ml
Canavalia ensformis 0.1 100 60 30
Ricinus communis 0.1 15 45 0
Saphora japonica 0.1 15 40 n.d.
Arachis hypogaea 0.1 65 100 n.d.
the amino acids cysteine, histidine and imidazole, decrease the
activity of both enzymes.
Inhibitory effects of lectins binding to mannosyl or
galactosyl residues may be caused by direct action on the
glycoprotein enzymes orland by indirect interference with
substrate recognition. If galactose residues are part of the
enzyme/substrate interaction with or in the cell wall, modifi-
cation or removal of this residue should then inhibit or dimin-
ish biological activity. Indeed, when weused galactose oxidase
specifically to oxidize, or a and P-galactosidase to remove this
sugar from sporangial walls, the P-galactose-modified cells
were totally inert as substrates in the biological assay; whereas
the a-galactosidase-treated cells completely retained their sub-
strate property. This points to a P-galactose residue as require-
ment for the recognition of the sporangial wall by the enzyme.
Enzymatic features of the autolysins
From the overall analysis of the C. reinhardtii cells walls
[31, 321, showing carbohydrates (mainly arabinose, galactose
and some mannose) and (hydroxyproline-rich) proteins as the
main components, lysing enzymes must be either proteases or
glycosidases. By following the purification of both sporangial
autolysin and gamete autolysin by assays for carbohydrates
[23], exoglycosidases [24] and peptides [25], we found that
only the peptidase activity increases essentially proportional
to the specific activity towards the respective natural cell wall
substrate, whereas the other activities were totally removed.
To prove the proteolytic nature of the enzymes we tested
various synthetic peptides or natural and denaturated proteins
as substrates. There are definite differences in the proteolytic
specificity of the two autolysins. The gamete autolysin splits
synthetic peptides in chymotryptic fashion: the hexapeptide
Leu-Trp-Met-Arg-Phe-Ala yields Leu-Trp-Met, Arg-Phe-
Ala, Leu-Trp and Met-Arg-Phe-Ala, as revealed by thin-layer
chromatography. In contrast, the sporangial autolysin does
490
Fig. 4. SDS gel electrophoresis of splitting products of the NaC104-
soluhlepart (fraction A ) ofthe zoospore cell wall with gamete autolysin.
Lane a: wall fragments were isolated from NaCI04-soluble extracts
of zoospore walls of strain 11 32 by column chromatography on
Sepharose 2B (for details see Materials and Methods); 50 Fg was
incubated with 25 U gamete autolysin of strain 137 cat 37C for 10 h,
lyophilized, solubilized in SDS-sample buffer and used for
electrophoresis. Lane b: same as a, but with EDTA-inhibited autolysin
(control I); lane c: 25 U gamete autolysin (control 11). Number indi-
cate molecular mass in kDa
not utilize this peptide. Neither enzyme accepts native proteins
such as haemoglobin, casein or bovine serum albumin, but
they do attack heat-denatured substrates which contain regu-
larly spaced proline or hydroxyproline residues like gelatin,
azocoll or heat-denaturated collagen. However, they do not
hydrolyze poly(pro1ine) or poly(hydroxypro1ine). The typical
substrate for collagenase A, Gly-Pro-Gly-Gly-Pro-Ala, is not
split by either the gamete or the sporangia autolysin.
Points of attack in the cell wall
To study the sites of attack of the autolysins we used
fractions of the natural cell wall substrate. The cell wall of C.
reinhardtii is composed of several layers [I 5,16,31] which can
be separated by chaotropic reagents such as NaC10, into a
soluble and an insoluble part. The NaC104-insoluble portion
represents the innermost layer [15, 161. Under natural con-
ditions the enzyme will work from the inside, i.e. the enzyme
released from inner vesicles will begin its lytic attack at the
layer of the wall closest to the cell body. But it is also able, at
least after formaldehyde-treatment of cells, to penetrate from
the outside, and wemake use of this in our assays. To gain
insight into its action, weisolated wall fragments by combin-
ing the method of Roberts et al. [15, 161 with that of
Salvendran [27, 281 by extracting first exhaustively with
NaClO,, then oxidatively with acidic NaC102. The three frac-
tions obtained (fraction A =NaC104-soluble, fraction B =
NaC1O4-inso1uble/NaClO2-soluble and fraction C =
NaC102-insoluble) were incubated separately with the
autolysins for 10 h, and then release of peptide fragments was
measured by the fluorescamine assay. Comparing the amount
of peptides released from these fractions with both autolysins
Fig. 5. SDS gel electrophoresis of splitting products qf the chlorite-
soluble part (fraction B) of sporangial cell walls with spurungiul
autolysin. Lane a: 200 pg chlorite-soluble material (fraction B) was
incubated with 100 U sporangial autolysin at 37C for 10 h,
lyophilized, solubilized in SDS-sample buffer and used for
electrophoresis. Lane b: same as a, but with EDTA-inhibited
autolysin. Numbers indicate molecular mass in kDa
wefound that fractions B and C are split at a considerably
higher rate than fraction A. From this weconclude that the
enzymes have a preference for the highly condensed and cross-
linked layers of the cell walls.
SDS-gel electrophoretic analysis was employed to follow
splitting of separated cell wall fragments. Column chro-
matography on Sepharose 6 B of fraction A of zoospore walls
of strain 11 32 aM gave an electrophoretically almost uniform
fragment of 64 kDa, which was split by gamete autolysin from
strain 137 c into multiple fragments showing a distinct triplet
pattern between 45 kDa and 37 kDa in Fig. 4.
On incubation of the not so easily disaggregated fraction
B of the sporangial wall of strain 77 81 with sporangial
autolysin of the same strain, four predominant new bands
occur with molecular masses of 23, 34, 64 and 150 kDa. Also
some less defined bands between 70 - 100 kDa can be detect-
ed, as seen from Fig. 5. This demonstrates the degradation of
the cross-linked high-molecular-mass wall structures.
SDS-gel electrophoresis of the split products of the densely
aggregated insoluble part (fraction C) of the sporangial wall
with the sporangial autolysin revealed products with mo-
lecular masses of 114,95,81,67 and 64 kDa (not shown). The
characteristic splitting points within the wall molecules are
still unknown.
DISCUSSION
The cell walls in C. reinhardtii are lysed by proteolytic
enzymes (autolysins) which are synthesized at characteristic
points during the developmental cycles of the alga (release of
zoospores or fusion of gametes). They have strong specificity
towards the respective wall structures. The sporangial en-
zymes of two related strains (77 81 and 11 32 aM) have similar
molecular properties and kinetic characteristics. They split
only sporangial walls. The gamete autolysin (strain 137 c), on
the other hand, seems to have a more general repertoire of
substrates, it even splits respective model peptides in a
chymotryptic mode. Both enzymes differ in molecular mass
and size, and in molecular characteristics, such as pH opti-
mum, model substrate specificity and relative sensitivity
towards inhibitors. But both are very specifically addressed
towards connective-tissue-like substrates. Their poly[(hy-
droxy)proline] specificity likens the autolysins remarkably to
collagen- and gelatin-splitting enzymes. In fact, both enzymes
are sensitive to inhibitors of collagenases like EDTA, cysteine,
o-phenathroline, phenylmethylsulfonyl fluoride and diiso-
propylfluorophosphate [33 - 361. But tests with non-de-
natured collagen show that the autolysins are not true collage-
nases: they do not split the intact triple helix of collagen,
but only attack heat-denatured collagen or gelatin. In this,
however, the cell wall autolysins are the first examples of
connective-tissue-splitting plant proteases, though they do not
attack typical scleroproteins of higher plants such as extensin
(from potatoes) or gliadin (from maize). Of the algal cell walls
they fragment the inner, insoluble layer with the highest rate,
but also the NaC104-soluble part. This may explain why the
in vitro assays with killed sporangia or zoospores work. The
characteristic splitting points, however, have yet to be eluci-
dated by end group analysis.
Our search for other hydrolytic activities in the crude
extracts showed that there are several glycosidic and at least
one other proteolytic activity, which are also secreted during
release of reproductive units. The glycosidic enzymes may be
responsible for dissolving the jelly inside the cells. Microscopic
observation (india ink contrast) with killed sporangia shows
that on prolonged incubation crude autolysin preparations
totally Free the zoospores from the accompanying jelly,
whereas purified autolysins quickly dissolve the wall proper,
leaving the naked zoospores still embedded in gelatinous ma-
terial. The function of the second proteolytic activity, a casein-
splitting protease, is not clear: it may transform the autolysins
from inactive proenzymes in the manner described for other
proteases. This caseinase, contained in crude extracts of spo-
rangial and gamete autolysin, can be separated from the auto-
lysins by ion-exchange chromatography; weassume that it was
as a contamination in our earlier preparations that wefound
some caseinolytic activity of the sporangial autolysin [9].
Wedo not know when the autolysins are synthesized and
whether or where they are stored as proenzyme or enzyme-
inhibitor complex. These questions can be approached by
using antibodies against the highly purified autolysins. The
signal of release of the gamete autolysin seems to be flagellar
contact of both mating types [4, 61. The messages for the
release of sporangia autolysin are low oxygen pressure and
illumination of a certain duration to liberate zoospores. It will
be interesting to know how this is transformed into biochemi-
cal action.
We thank Dr Y. Matsuda (Amsterdam) and Prof. Dr U. G.
Schlosser (Gottingen) for providing methodological council and the
algal strains used in this study; Dr F. Klis and Dr A. Musgrave
(Amsterdam) and Dr K. Roberts (Norwich) for encouraging dis-
cussions and advice, and Dr B. c. Adelmann-Grill (Martinsried) for
help with the collagenase assays. The technical assiatance of Mrs E.
Glees is gratefully acknowledged. Financial support by the Deutsche
Forschungsgemeinschaft (Bonn) through J a 58/22 and the Fonds der
Biologischen Chemie (Frankfurt) made this study possible.
49 1
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