ARCHIVES
OF BIOCHEMISTRY
Vol. 295, No. 2, June,
AND
BIOPHYSICS
pp. 273-279,1992
A Molecular and Biochemical Analysis of the Structure
of the Cyanogenic ,&Glucosidase (Linamarase) from
Cassava (Aknihot esculenta Cranz)
Monica A. Hughes,’
Kate Brown,
Adi Pancoro,
B. Stuart Murray,
Elli Oxtoby,
and Jane Hughes
Department of Biochemistry and Genetics, The University, Newcastle upon Tyne NE2 4HH, United Kingdom
Received
November
20, 1991, and in revised
form
February
3, 1992
The cyanogenic
&glucosidase
(linamarase)
of cassava
is responsible
for the first step in the sequential
breakdown of two related cyanoglucosides.
Hydrolysis
of these
cyanoglucosides
occurs following
tissue damage and leads
to the production
of hydrocyanic
acid. This mechanism
is widely regarded
as a defense mechanism
against predation.
A linamarase
cDNA clone (pCAS5)
was isolated
from a cotyledon
cDNA library
using a white clover /3glucosidase
heterologous
probe. The nucleotide
and derived amino acid sequence is reported
and five putative
N-asparagine
glycosylation
sites are identified.
Concanavalin
A aflinity
chromatography
and endoglycosidase
H digestion
demonstrate
that linamarase
from cassava
is glycosylated,
having
high-mannose-type
N-asparagine-linked
oligosaccharides.
Consistent
with this structure and the extracellular
location
of the active enzyme
is the identification
of an N-terminal
signal peptide
on
the deduced amino acid sequence of pCAS6.
o lssz ACMI~IOIC
Press,
Inc.
Cassava (Munihot es&en&z Cranz) is an important
tropical root crop (1,2); however, the plant is cyanogenic
(that is, hydrocyanic acid is released from damaged tissue)
and this has been recognized as a potential health hazard
to consumers of the crop (3, 4). Both the leaves and the
roots of cassava are cyanogenic and, although both are
eaten, cassava’s importance as a crop is due to the large
tuberous roots which are a staple carbohydrate source for
many communities in the tropics (1,2). Hydrocyanic acid
is produced following mechanical damage to tissues. This
tissue damage exposestwo structurally related cyanogenic
glucosides (lotaustralin and linamarin) to the sequential
action of two enzymes, a &glucosidase and a nitrilase (5,
6). Cassava; white clover, Trifolium repens L. (7); flax,
’ To whom
correspondence
should
0003.9861/92
$5.00
Copyright
0 1992 by Academic
Press,
All rights
of reproduction
in any form
be addressed.
Linum usitutissimum L. (8); Lotus species (9); rubber,
Hevea braziliensis L. (10); and lima bean, Phaseolus lunatus L. (ll), all produce the same cyanoglucosides and release hydrocyanic acid from damaged tissue following hydrolysis of these glucosides. White clover and birds-foot
trefoil (Lotus corniculutus) are polymorphic for the cyanogenic character, with stable acyanogenic plants existing in wild populations of both species (12). The biochemical and genetic basis of the acyanogenic phenotype
has been extensively studied in white clover (13-15) and
the cyanogenic ,&glucosidase (linamarase) has been cloned
from this species (16).
There have been a number of recent studies of the purification and kinetics of the cyanogenic fl-glucosidase
(linamarase) from cassava (17-19), and in addition it has
been reported to be located in the cell walls of cassava
leaf tissue (18). In this paper we report the isolation of a
cassava linamarase cDNA clone from a cotyledon cDNA
library using a fragment of a white clover P-glucosidase
cDNA clone as a heterologous probe. Analysis of the glycosylation of the active cassava linamarase is reported
together with the identification of potential glycosylation
sites on the deduced amino acid sequence.
MATERIALS
AND
METHODS
Growth of seedlings.
Cassava seeds collected from the plant CM122311 were supplied
by Dr. C. Hershey,
CIAT, Cali, Colombia.
They were
germinated
in the dark on damp vermiculite
in a 25”C/37”C,
12-h cycle
until the radicle emerged. They were then maintained
at 25°C and given
light when the cotyledons
had fully expanded.
Enzyme purification
and assays.
Linamarase
from young leaves of
white clover was purified
as described
in Hughes and Dunn (15). Linamarase was extracted
from acetone powder
(1.5 g) of young cassava
leaves by soaking overnight
in 15 ml 0.4 M Tris-maleate
buffer, pH 5.6,
at 4°C. The slurry was squeezed through
a nylon mesh and centrifuged
at 20,OOOg for 20 min. This crude extract
(12 ml) was fractionated
by
molecular
exclusion chromatography
using Sephacryl
S-300 (Pharmacia)
(80 X 2.5 cm) and 0.2 M Tris-maleate
buffer, pH 5.6. The active peak
from five molecular
exclusion
columns
was diluted to 0.05 M Tris-maleate, pH 5.6, and adsorbed
onto a DEAE-Sepharose
(8.0 X 5.0 cm)
273
Inc.
reserved.
274
HUGHES
ET
AL.
1.3
1.2
1.1
1 .o
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
FRACTION
q
FIG. 1. Concanavalin
A affinity
with 0.2 M Na phosphate
buffer,
ethanediol.
CLOVER
&
NU’lBER
CASSAVA
chromatography
of the cyanogenic
fi-glucosidase
(linamarase)
from cassava and white clover. Columns washed
pH 7.4, containing
1 M NaC1; (A) 0.1 M a-methyl
mannoside;
(B) 1.0 M a-methyl
mannoside;
(C) 50% v/v
(Pharmacia)
ion-exchange
column.
Protein
was eluted using a 0.05 to
0.4 M Tris-maleate,
pH 5.6, gradient
and the active peak (eluting
at
0.26 M) concentrated
using a Diaflo YM30 filter (Amicon).
Concanavalin
A affinity
chromatography
and linamarase
assays were carried out as
in Hughes and Dunn (15).
Endoglycosidase
digestions.
Endoglycosidase
D (endo-P-D-acetylglucosaminidase
D) (1 mu) or endoglycosidase
H (endo+-N-acetylglycosaminidase H) (1 mu) (Boehringer-Mannheim
GmbH)
was used to digest
5 pg of linamarase
(final concentration
0.25 rg/pl).
Prior to digestion,
linamarase
from both species was denatured
by the addition
of SDS’ to
0.5% and 2-mercaptoethanol
to 0.025 M and then heating in a boiling
water bath for 2 min. The endoglycosidase
digestions
were carried out at
37°C for 20 h. Proteins
were analyzed using 10% SDS-PAGE
(20).
Preparation
of P-glwosidase
forpeptide
sequencing.
Purified
cassava
linamarase
at a concentration
of 0.5 mg/ml in 0.1 M Tris-maleate,
pH
5.6, and 0.5% SDS was boiled for 3 min and then digested for 1 h at
37°C with 30 pg/ml a-chymotrypsin.
Aliquots
(100 ~1) were boiled for
2 min with an equal volume
of SDS-PAGE
sample buffer containing
0.125 M Tris-HCl,
pH 6.8,4% SDS, 20% glycerol, 5% 2-mercaptoethanol,
and 0.002% bromophenol
blue. The samples were run on an 11.5% SDSPAGE (20) for 4f h at 40 mA.
The separated peptides were electroblotted
onto a Problott
membrane
for 24 h at 100 V in transfer
buffer consisting
of 48 mM Tris, 39 mM
glycine, 10% methanol,
and 0.03% SDS. The blot was stained for 2 min
in 0.1% Coomassie
brilliant
blue R and 50% methanol,
and destained
for 10 min in 50% methanol
and 10% acetic acid. Excised bands from
the dried membrane
were sequenced
by Dr. Kathryn
Lilley,
Protein
Sequencing
Facility,
Biochemistry
Department,
Leicester
University.
umns with the Pharmacia
mRNA
purification
kit. First-strand
cDNA
synthesis
was catalyzed
by Moloney
murine
leukemia
virus reverse
transcriptase
and the second-strand
synthesis used RNase H degradation
of the RNA:cDNA
duplex followed by DNA polymerase
I-catalyzed
synthesis, using the Pharmacia
cDNA synthesis kit. EcoRI/NotI
Pharmacia
adaptors
were added to blunt-ended
cDNA molecules
using the Pharmacia cDNA synthesis
kit protocol.
The resulting
EcoRI-terminated
cDNA molecules
were phosphorylated
with T4 polynucleotide
kinase
and ligated into EcoRI-cut,
dephosphorylated
X&10.
Phage in vitro
packaging
was carried out as in Sambrook
et al. (22) and serial dilutions
were plated on the selective host Escherichia
coli NM514 and nonselective
host L87 in order to estimate
the proportion
of recombinant
clones in
the resulting
library.
DNA from approximately
3000 plaques was transferred to duplicate
nitrocellulose
filters. Radiolabeled
probe DNA was
made using the Boehringer-Mannheim
GmbH
random prime DNA labeling kit. Hybridization
of the filters with labeled DNA was carried
out as described
in Sambrook
et al. (22) with the final wash being 2 X
SSC (1 X SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) and 0.1%
SDS for 20 min at 50°C. Plaques which gave a positive signal on duplicate
filters were purified,
digested with Not1 to determine
insert size, and
subcloned
into the NoB site of pBluescript
KS +/(Stratagene).
DNA
sequencing
and computer
analysis.
Known
restriction
fragments were subcloned
into the sequencing
vector Ml3 mp18. DNA sequences were determined
in both directions
using Sequenase
version
2.0 (United
States Biochemical
Corp.) and synthetic
oligonucleotides.
DNA and amino acid sequences were analyzed
using the DNAsis
and
Prosis software
from Pharmacia.
The rules for equivalence
of amino
acids used are as follows: (A, G); (T, S); (R, K); (L, I, V); (Y, F), (E, D);
(N, Q).
Construction
and selection of cDNA clones.
Total RNA was isolated
from yellow cotyledons
using the method of Hughes and Pearce (21).
Poly(A)+
RNA was purified
by affinity
chromatography
using spun col-
RESULTS
’ Abbreviations
used: SDS, sodium
amide gel electrophoresis.
Figure 1 shows the results of affinity chromatography
of purified cyanogenic P-glucosidase
(linamarase)
from
white clover and cassava using the lectin, concanavalin
dodecyl
sulfate;
PAGE,
polyacryl-
STRUCTURE
OF
THE
CYANOGENIC
A, immobilized on Sepharose 4B beads. Concanavalin
A
has a high affinity for high-mannose
oligosaccharides
(23)
and will bind to glycoproteins
which contain these structures. This figure shows that all the applied enzyme binds
to these columns so that no activity was detected in the
wash. Separate, but identical, columns were used for each
enzyme. Both enzymes are eluted with a-methyl
mannoside but elution of the cassava enzyme requires 10 times
the concentration
(1.0 M methyl mannoside) required to
elute the white clover enzyme (0.1 M methyl mannoside).
These data show that the cassava linamarase is similar
to white clover linamarase, being a glycoprotein
containing high-mannose-type
oligosaccharides.
In order to investigate the oligosaccharide
composition
of the cassava linamarase in more detail, the enzyme was
purified from leaf tissues to a single Coomassie blue SDSPAGE band and aliquots of this preparation were digested
with endoglycosidase
D and endoglycosidase
H. Figure 2
shows the results of endoglycosidase
H digestion on the
relative molecular mass of cassava linamarase as measured by SDS-PAGE.
Purified white clover linamarase
is included for comparison.
Analysis of the relative mobility of the enzyme preparations
and markers in a number
of gels indicates that the native cassava linamarase has
a relative molecular mass of 70,000 which is reduced to
65,000 by endoglycosidase
H digestion. The white clover
linamarase is confirmed as 62,000 M, (24) which is reduced
to 59,000 M, by endoglycosidase
H digestion. This technique indicates that the proportion of N-linked oligosaccharide in the native enzyme is 7.2% in cassava and 4.8%
in white clover. Endoglycosidase
D had no effect on the
size of these enzymes and was considered to be inactive
against the oligosaccharides
of both the cassava and the
white clover linamarase.
Endoglycosidase
D can only digest high-mannose
oligosaccharides
of the M5 type, whereas endoglycosidase
H cleaves M5, M8, and M9 high-mannose
and hybrid
structures
(25-28). Thus the high affinity of concanavalin
A and the endoglycosidase
H digestion show that cassava
produces a cyanogenic P-glucosidase which is glycosylated
with asparagine-linked
M8 or M9 high-mannose
oligosaccharides. The estimation of the relative molecular mass
of proteins by SDS-PAGE
must be interpreted with caution. First, glycoproteins
are known to give anomalous
data (29) and, second, basic proteins have been shown to
give to an overestimate of M, in SDS-PAGE
(30). In both
cases the phenomenon is due to nonstochiometric
binding
of SDS to the proteins.
Figure 3 shows the linamarase specific activity of cassava seedlings during the first 12 days of germination.
It
can be seen that there is a rapid increase in enzyme activity during germination,
with most of the enzyme associated with the aerial parts (primarily
hypocotyl and
cotyledons)
of the plant. The cotyledons became auxotrophic (photosynthetic)
between Days 10 and 11 and true
leaves began to expand after Day 11. A cDNA library was
P-GLUCOSIDASE
FROM
1
275
CASSAVA
2
3
4
5
6
92
6%
45
29
FIG. 2.
SDS-PAGE
of purified cyanogenic
fl-glucosidase
(linamarase)
from cassava and white clover: Effect of endoglycosidase
H on relative
molecular
mass. Well 1, molecular
weight markers,
&f, X 10e3; well 2,
endoglycosidase
H digestion of white clover linamarase;
well 3, untreated
white clover linamarase;
well 4, endoglycosidase
H; well 5, endoglycosidase H digestion
of cassava linamarase;
well 6, untreated
cassava linamarase.
made in the X vector gtl0, using EcoRI/NotI
adaptors,
from mRNA extracted from lo-day-old
cotyledons. The
library was screened with the 704-base SspI fragment of
the white clover /3-glucosidase cDNA clone pTRE361 (16).
This fragment has been shown to include a region conserved among a number of P-glucosidases (16). Six clones
were recovered and subcloned into the sequencing vector
Ml3 mp18 and the plasmid Bluescript KS.
Figure 4 shows the nucleotide and deduced amino acid
sequence of one of the longest selected clones (pCAS5).
This clone hybridizes
to a leaf mRNA which is 1.8 K
bases long (data not shown) and the clone is considered
to be virtually
full length. There is no poly(A) tail on
pCAS5 but a putative polyadenylation
signal is present
between bases 1650 and 1662. The native cassava linamarase purified from young leaf tissue is blocked at
the N-terminus.
The amino acid sequences derived from
two internal peptides, which were generated by digestion with cu-chymotrypsin,
are present in the pCAS5derived amino acid sequence and are underlined in Fig.
4. The identification
of these sequences confirms
the
identity of clone pCAS5 as the cyanogenic /3-glucosidase
(linamarase).
The derived amino acid sequence of pCAS5 has considerable homology with that of the white clover linamarase cDNA clone, pTRE104 (16). In the region residue
44 to residue 354 (310 amino acids), 66% of the amino
acids are either identical or equivalent in the white clover
and cassava sequences (54% identical) (Fig. 5). Significant
homology also exists within this region between cassava
linamarase and the white clover noncyanogenic
P-glucosidase, pTRE361
(16), with 50% of the amino acids
276
HUGHES
ET
AL.
60.00
0
2
4
o
FIG.
3.
Specific
activity
of the cyanogenic
fl-glucosidase
SHOOTS
(linamarase)
either identical or equivalent (43% identical); however,
this homology requires the insertion of a two-amino-acid
gap in the white clover fl-glucosidase
sequence. The five
putative iV-asparagine glycosylation
sites on the cassava
linamarase (31) have been boxed on Fig. 4. They all lie
in the C-terminal
region of the protein and only one glycosylation site (NAT; residues 368-370) is conserved in
the white clover and cassava linamarase sequences. The
glycosylation
of this site in the cassava enzyme is problematic since a proline residue exists adjacent to the consensus glycosylation
motif (NX[ST])
and this is thought
to exclude such a site (31). If this glycosylation
site is not
included in cassava linamarase, both the white clover and
the cassava proteins have four potential glycosylation
sites, although they are distributed
in very different regions of each sequence.
Figure 6 shows a hydrophobicity
plot of the deduced
amino acid sequence of cassava linamarase
(32). This
analysis shows a prominent hydrophobic region at the Nterminus. It is known that the white clover linamarase
has an N-terminal
peptide cleaved during cotranslational
processing (16). Since both enzymes have been shown to
have an extracellular
location in leaf tissue (18, 33) and
since N-asparagine
glycosylation occurs within the lumen
of the endoplasmic
reticulum
(34, 35) a similar signal
peptide may be expected in cassava. The N-terminal
amino acid sequence of cassava linamarase is not known
but the hydrophobicity
plot (Fig. 6) would predict that
the active enzyme begins at residue 12. The deduced relative molecular mass of the resulting polypeptide is about
62,000 Mr.
6
s
10
12
DAYS
+
ROOTS
in the shoots
(cotyledons
and hypocotyl)
and roots of germinating
cassava
DISCUSSION
The amino acid sequence deduced from the cassava linamarase cDNA clone pCAS5 reveals details of a structure
which can be predicted from the demonstrated
extracellular location of the enzyme (18). Six cDNA clones were
isolated from the cassava cDNA library, five of these are
judged by restriction
site maps to represent the same
mRNA sequence. One other member of this group of
clones (pCAS6) has been sequenced. It is 15 bases longer
then pCAS5 at the 5’ end but the rest of the sequence
confirms that determined for pCAS5, including the unusual five GAT repeats at position 78. The amino acid
composition of the sequence predicted by pCAS5 is most
similar to the major petiole ~14.3 isoform of linamarase
reported by Eksittikul
and Chulavatnatol
(17). The existence of isoelectric point variation in cassava linamarase
was also reported by Mkpong et al. (18) and the possibility
of variation existing at the primary level is suggested in
this study by the isolation of one cDNA clone which has
an extra restriction
site.
The signal hypothesis
proposed by Blobel and Dobberstein (36) is now widely accepted. The first step in the
secretory process of proteins is sequestration
into the endoplasmic reticulum (37,38) and this is usually associated
with the presence of an amino terminal signal sequence.
Such sequences are typically 13-30 amino acids long (39)
and have a core of at least nine hydrophobic residues. The
predicted signal peptide of cassava linamarase is rather
short (12 amino acids) but it is strongly hydrophobic, with
a mean hydrophobicity
index of +3.71 (32). It also has a
STRUCTURE
I
I
FIG. 4.
underlined,
Nucleotide
putative
OF
THE
CYANOGENIC
,&GLUCOSIDASE
FROM
CASSAVA
277
AC MC TTT CTT CA6 CTA TCA GGG ATG CTC GTC TTG TTC ATA AGC TTG TTG GCT CTC ACT AGG CCC,GCA ATG GGA ACT
RlVLFISLLAlTAPANGT
17
IS
78 GAT GAT GAT GAT GAT AAT ATT CCT GAC GAT TTT AGC CGT AA1 TAT TTT CCA GAT GAC TTC ATT TTT GGA ACG GCT ACT
19 0 0 D II II N I P 0 D F 5 A K V F P D D F I F G T AT
155
44
156 TCT GCT TAT CAG ATC GA1 GGT GAA GCA ACC GCA AA6 G6T AGA 6CA CCT AGT GTT TGG GAC ATA TTT TCC AAG GAG ACT
45 S A V 4 I E G E A T A K G A A P S V V D I F S K E T
233
70
234 CCA GAT AGA ATA TTA GAT GGC AGC AAT GGA GAC GTT GCA GTT GA1 TTC TAT AAC CGC TIC ATA CAA GAT ATA AAA AAC
71 P 0 R I LD
G S N G 0 V A V 0 F V N R V I4
0 I K N
311
96
312 GTC AA1 AAG ATG GGT TTT AAT GCA TTT AGA ATG TCC ATT TCA TGG TCT AGA GTT ATA CCA TCC 661, AGG AGA CGT GAA
97 V K K M G F N A F R N S I S M S A V I P S G R R R E
369
122
390 6GA GTG AAC GAG GAA 661, ATT CIA TTC TAC AAT GAT GTT ATC AAT GAA ATT ATA ICC AAT 661, CTA GAG CCT TTT GTT
123 G V N E E G 16
F V N D V I N E I I S N GLE
P F V
467
146
468 ACT ATT TTT CAT TGG GAT ACT CCT CAA GCA CT6 CAG GAC AAA TAT GGT GGC TTC TTA AGC CGT GAT ATT GTG TIC GAT
149 T I F H W D T P Q A LG
D K V G G F L S R 0 I V V D
545
174
546 TAT CTC CIA TAT GCA GAT CTT CTC TTT 6AA AGA TTC GGT GAT CGA GTG AAA CCC TGG ATG ACT TTT AAT 6AA CCA TCA
175 Y L 4 V A D LLF
E R F G 0 A V K P W N T F N E P S
623
200
624 GCA TAT GTT CGA TTT GCC CAT GAT 6AT 661, GTT TTT GCC CCT GGT CGA TGC TCA TCT TGG GTG AAT CGC CIA TGC CTA
2011
V V G F A H D D G V F A P G R C S S V V N A G CL
701
226
702 GCT GGA 6AC TCA GCC ICI GAA CCT TAT ATA GTT GCC CAT AAT TTG CTT CTT TCT CAT GCT GCA GCT GTT CAC CAA TAT
227 A G 0 S A T E P V I V A H N LLLS
H A A A V H P V
779
252
780 AGA All TAT TAT CAG G6A ACT CIA AA6 GGC AA6 ATT GGGATT ICC CTC TTT ACC TTC TGG TAT GAA CCT CTC TCC GAC
253 A K V Y P G T 4 K G K I G I T L F T F W V E P LS
D
857
276
859 AGT AAA GTT GAT GTG CIA GCA GCC AAA ACA CCC TTA GAT TTC ATG TTT GGA TTG TGG ATG GAT CCC ATG ACT TAT GGA
279 S K V D V 6 A A K T A LD
F M F G LW
N 0 P M T V G
935
304
936 CGA TAT CCA AGA ACT ATG GTA GAT TTA GCC GGA GAT AAA TTG ATT GGA TTT ACA GAT GAA GAA TCT CIA TTA CTT AGG
305 R V P R T M V D LA
G D K L I G F T 0 E E S 6 LL
R
1013
330
1014 GGA TCA TAT GAT TTT GTT GGA TTA CAA TIC TAC ACT GCA TAT TAT GCA 611, CCA ATT CCT CCA GTT GAT CCA AAA TTT
331G
S Y D F V G LQ
V V T A V V A E P I P P V 0 P K F
1091
356
1092 CGT AGA TIC AAA ACT GAT AGT GGT GTT AAT 6CG ACT CCT TAC GAT CTT AAT GOT AAT CTT ATT GGT CCA CAG GCT TAC
357RRYKTDS6V[~~PVDtNGNtIGP4AV
1169
362
I 170 TCG TCA TGG TTT TAC ATT TTT CCA AAA GOT ATT CGA CAC TTT TTG AAC TAT ACC AAA OAT ACA TAT AAT GAT CCA GTC
383 S S Y F V I F P K G I R H F L[YfJK
0 T V N D P V
1241
408
1248 ATT TAC GTT ACT GAG AAT GGGGTT 6AC AAC TAC AAT AAT GAA TCT CIA CCA ATT GAA GAG GCA CTT CA1 OAT OAT TTC
409 I Y V T E N G V 0 N V N[mlQ
P I E E A L 4 0 D F
1325
434
1325 AGG ATT TCG TIC TAT AAA AAG CAT ATG TGG AAT GCA CTA 661 TCT CTC AAG AAC TIC GGT GTT AAA CTC AAA GGT TAT
435 R I S Y V K K H H Y N A L G S L K N V G V K L K G V
1403
460
1404 TTT GCA TGG TCA TAT TTA GAC AAC TTC GAA TGG AAT ATT G6T TAT ACA TCA AGA TTT GGGTT6 TAC TAT GTA GAC TAC
461 F A W S V L D N F E W N I G V T S R F GLY
V V D Y
1481
486
1482 AAA AAT AAC CTA ACA A66 TAT CCC AAG AAA TCG GCT CAT T6G TTC ACA AAA TTC CTG AAT ATA TCG GTT AAT GCA AAT
497 K N (Hml
R Y P K K S A H Y F T K F L[-[V
N A N
1559
512
1560 AAT ATC TAT GAG CTT ACA TCA AAG GAT TCA AGG AAG 6TT GGC AAA TTC TAT GTG ATG TAG ATT ATG TCT GGA TGT TTT
513 N I V E LT
S K 0 S R K V G K F V V N t
1637
532
1538 GTG TGT ATC TCA TAA TTA AAT AAT ATC GTT GGG CIA TTA T6A AGC TCC AAT GAT CTA GCA TAT GTT GT
1705
and deduced
N-glycosylation
amino acid sequence of cassava cDNA clone
sites are boxed, a putative
polyadenylation
pCAS5. Amino acids
signal is underlined.
identical
to known
peptide
sequences
are
278
HUGHES
helix breaking proline residue at +2 and a threonine residue at -1 of the predicted cleavage site. Such residues
are commonly found in these positions relative to the signal peptide cleavage site (39).
In white clover, where the N-terminal
amino acid sequence of the active enzyme is known, an immunoprecipitable in vitro translation
product has been shown to
be processed by dog pancreas microsomes
(24). The antibiotic, tunicamycin, which prevents N-acetyl-asparagine
glycosylation,
has also been shown to inhibit synthesis of
the white clover linamarase. Precursor high-mannose
oligosaccharides
are known to be attached to asparagine
residues in proteins by an oligosaccharyltransferase
found
within the rough endoplasmic reticulum (38). Modification of the precursor
high-mannose
oligosaccharide
structure
also occurs in the endoplasmic
reticulum. Although not all secretory proteins have N-linked oligosaccharides, it is a feature of many. Glycosylation
is also
associated with the resistance of proteins to proteolytic
degradation. Its role in the cellular biology of cyanogenesis
in cassava may include a function both in sorting linamarase to the extracellular
matrix and in the enzymes
unusually high stability (19). The difference between the
predicted size of the unglycosylated
protein (62,000 M,)
and the size of the deglycosylated
protein (65,000 M,)
may be due to incomplete digestion by endoglycosidase
H, to an anomaly in size estimation using SDS-PAGE,
or to the presence of some 0-glycosidically
linked carbohydrates. The higher concentrations
of a-methyl mannoside required to elute the bound cassava enzyme from
concanavalin A-Sepharose
may indicate that it has more
oligosaccharide
residues compared with the white clover
linamarase. Thus both concanavalin
A affinity chromatography and endoglycosidase
H digestion are consistent
with the conclusion that the cassava linamarase has more
ET
AL.
e
clover
cassava
;y
110
sequence:
30
sequence:
FAPGFVFGTA
I
44 FPDDFI
DTFTRKYPEK
III
IKDRTNGDVA
ll~lll
III’I I’ I
GTA TSAYQIEGEA
IDEYRRYKED
IIIIIIIIIIIIIl’I
ILMSNGDVA
DIFSKETPDR
SSAFQYEGM
VDFYNRYIQD
VLPKGKLSGG
VNREGINYYN
NLINEVLAN
160
RGFLGRXIVD
DFRDYAELCF
KEFGDRVKRW
174
"'
' "
GGFLSRDIVY
"
'I"
DYLQYADLLF
210
PGRCSDWLKL
NCTGGDSGRE
224
PGRCSSWVNR
QCLAGDSATE
260
IGITLVSRWF
EPASKEKMV
274
IIIII I II EPLSDSKVDV
‘I I I II
IGITLFTFWY
III lll’l
QAAKTALDFM
310
VRKRLPKFST
BESKELTGSF
DFLGLNYYSS
IGIHKDRNLD
II IKNVKKNII
FETJGKGPSIW
I IIIII
AYRFSISWPR
III IIll
GFN AFl3NSISWSR
I
124
324
IIIII
II
II II
AGDKLIGFTD
' ER 6Li?u
PYLAARYQLL
ITLNEPWGVS
MNAYAYGTFA
dF&dYV
GFjrHDdd!
ARAAAMU
I I I I I I PYIVAHNUL
I I I I I I I SHAAAVHQYR
II II
DAAKRGLDFU
LGWPWHPLTX
KYYQGTQKGK
GRYPESURYL
I I I I GRYPRTMVDL
IIII I’ I
FGLWHDF'HTY
III I III DFVGLQYYTA
IIIIIIlll
EESQLLRGSY
FIG.
5. Homology
of the deduced amino acid sequence 30 to 340 of
white clover lmamarase
(pTREl04)
and the deduced amino acid sequence
44 to 354 of cassava linamarase
(pCAS5).
Amino acids which are either
identical
or equivalent
are marked
with a bar.
I
318
I
424
of the deduced
amino
I
532
acid sequence
of
oligosaccharide
residues compared with linamarase from
white clover and, since they have the same number of
potential glycosylation
sites, this implies that, at least in
white clover, not all of these potential sites are glycosylated.
ACKNOWLEDGMENTS
This work was supported
by the UK Overseas
Development
Agency
(NRI Extramural
Contract
X0156),
The Rockefeller
Foundation,
and
the Commission
of the European
Communities
program,
Science and
Technology
for Development.
The authors
thank Dr. A. R. Hawkins
and the Wellcome
Trust for the synthetic
oligonucleotides.
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