Note: Descriptions are shown in the official language in which they were submitted.
21 70355
Translation from German
Reference No. 2-23b/sg
_______________________
WORLD INTELLECTUAL PROPERTY ORGANIZATION
PCT [Logo]
~ ~ International Bureau
o u)
~ ~nNATIoNAL APPLICATION PUBLISHED UNDER THE
~ ~ lh~ ATIoNAL PATENT COOPERATION TREATY (PCT)
Z N
YN (51) International Patent (11) International
Classification6: A2 Publication Number:
3 ~ C12N 15/31, CO7K 14/37, WO 95/06122
~ I C12N 1/21, 15/62,
Z A61K 39/35, GOlN 33/569 (43) International
.~ Publication Date:
~ ~ March 2, 1995
c L~ ( 02.03.95)
C ~ (21) International file No.: PCT/AT94/00121
(22) International application date: Aug. 24, 1994 (24.08.94)
~L ( 30) Priority data:
A 1726/93 Aug. 27, 1993 (27.08.93) AT
(71) Applicant (for all designated states other than US): BIOMAY
PRODUKTIONS- UND HANDELSGESELLSCHAFT M.B.H. [AT/AT]; Herrenstrasse
2, A-4020 Linz (AT).
(72) Inventor; and
(75) Inventor/Applicant (only for USJ;
ACHATZ, Gernot [AT/AT], schie~startstrasse 7/III/7, A-5020
Salzburg (AT); OBERKOFLER, Hannes [AT/AT]. A-5732 Muhlbach 98
(AT); SIMON, Birgit [AT/AT], Dirnbockweg 17, A-8700 Leoben (AT);
~ ~ UNGER, Andrea [AT/AT], Zaisberg 14, A-5201 Seekirchen (AT);
p ~ LECHENAUER, Erich [AT/AT], Dottlstrasse 16, A-5400 Hallein (AT);
HIRSCHWEHR, Reinhold [AT/AT], Nauseagasse 18/10, A-1160 Vienna
(AT); EBNER, Christoph [AT/AT], St. Elisabethplatz 4/13, A-lo4o
Vienna (AT); KRAFT, Dietrich [AT/AT], Montigasse 1, A-1170 Vienna
(AT); PRILLINGER, Hans-Jorg [AT/AT], Ebersbrunn 70, A-3711
Ebersbrunn (AT); BREITFNRAr~, Michael [AT/AT], Alfred
Kubinstrasse 11/11, A-5020 Salzburg (AT).
(74) Attorneys: ITZE, Peter etc., Amerlingstrasse 8, A-lo6l Vienna
(AT)
(81) Designated state~: AU, CA, FI, JP, NO, US, European Patent (AT,
BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE)
Published
Without international search report, and to be republished upon
receipt of that report.
- 21 70355
.
(54) Title: Recombinant Alternaria alternata allergens
(57~ Abstract:
The invention concerns recombinant DNA molecules which code for
polypeptides possessing the antigenicity of the allergens Alta53~
Alta22 and Altall, or for peptides having at least one epitope of
these allergens. These molecules are characterized in that they
contain nucleic acid sequences which correspond in homologous
fashion to the sequences 1, 3-5, 7-9, 12 and 13 or to parts of
these sequences, or nucleic acid seqUenCes which hybridize with
the above nucleic acid sequences under strictly controlled
conditions.
FOR INFORMA TION ONL Y
Codes used to identify States party to the PCT on the front pages of
documents which publish international applications under the PCT.
AT Austria GA Gabon MR Mauritania
AU Australia GB United Kingdom MW Malawi
BB Barbados GE Georgia NE Niger
BE Belgium GN Guinea NL Netherlands
BF Burkina Faso GR Greece NO Norway
BG Bulgaria HU Hungary NZ New Zealand
BJ Benin IE Ireland PL Poland
BR Brazil IT Italy PT Portuga
BY Belarus JP Japan RO Romania
CA Canada KE Kenya RU Russian Federation
CF central African KG Kirgistan SD Sudan
Republic
CG Congo KP North Korea SE sweden
CH switzerland KR south Korea SI slovenia
CI Ivory Coast KZ Kazakhstan SK slovakia
CM Cameroun LI Liechtenstein SN Senegal
CN china LK Sri Lanka TD chad
CS Czechoslovakia LU Luxembourg TG Togo
CZ Czech Republic LV Latvia TJ Tadzhikistan
DE Germany MC Monaco TT Trinidad & Tobago
DR Denmark MD Moldavian Rep. UA Ukraine
ES Spain MG Madagascar US United States of
America
FI Finland ML Mali UZ Uzbekistan
FR France MN Mongolia VN Vietnam
21 70355
.
-
WO 95/06122 PCT/AT94/00121
Recombinant Alternaria alternata allerqens
The present invention relates to recombinant DNA moleculesthat code for polypeptides which possess the antigenicity of
the allergens Alta53, Alta22 and Altall, or for peptides
which have at least one epitope of these allergens.
The aforementioned allergens of Alternaria alternata, as
well as the peptide sequences derived from the primary cDNA
sequences of these allergens lead, in patients allergic to
fungi, to a pathologic immune response with overshooting of
IgE antibodies. Recombinant allergens or partial peptides
having an immunogenic action may be used not only for
improving the diagnosis but also for in vivo or in vitro
induction of immunotolerance or anergy of T lymphocytes.
In the last few years epidemiological studies have shown
that allergies are becoming increasingly frequent. Allergic
diseases have many causes. Today the concept of allergens
such as pollen, animal hair and excreta of household dust
mites is probably familiar to everyone (Wuthrich 1991,
Miyamoto 1992). However, for professionals active in this
field, molds still leave many unanswered questions with
regard to their biologic and allergologic significance. Not
least, it is their very great variability and adaptability
to different living conditions that make research with these
fungi difficult. 98% of known fungi are country dwellers.
For most fungi, climatic conditions of 80% atmospheric
humidity and temperatures of 20C represent ideal living and
propagation conditions.
21 70~55
,
The mechanisms of mold allergies are not known precisely,
and seem more complex and complicated than in the case of
the usual immediate-type inhalation allergies. The
possibilities of sensitization via the digestive tract and
not only via the respiratory tract are being discussed and
are the subject of intensive research.
Immediate-type allergies (Type I allergy) are induced by IgE
antibodies, which contact effector cells (mast cells of the
mucosal and connective-tissue type as well as blood
basophilic granulocytes) with their Fc part via receptors,
and upon contact with allergen, cause the release of
inflammatory substances (histamine, heparin, arachidonic
acid metabolites, etc.) (Roitt 1991, Klein 1990). The
formation of such IgE antibodies takes place through B
lymphocytes, which are stimulated by soluble substances
(lymphokines) that are secreted by activated T lymphocytes
to secrete the antibodies (Parronchi et al., 1991).
Every immune response is started off by cells which actively
phagocytize the antigen present. For this reason these
cells are also called "phagocytes." They are dendritic
cells, but also monocytes which are differentiated to
macrophages at a later stage. All these cells have the
capacity of DIAPEDESIS, which enables them to leave the
blood system and penetrate into body cavities, etc. The
main part of antigen ~nni h;lation is carried out by the
macrophages. After phagocytosis they decompose the antigen
to highly immunogenic peptides (average size about 15 amino
acids) and, together with the MHC (major
histocompatibility)-proteins expressed on the macrophages,
present them to the T lymphocytes. The central role played
at this point by the T lymphocytes should be emphasized.
Only at this point of the immune response can it be
distinguished -- and this is the central role of the
21 70355
T lymphocytes -- whether the antigen presented is "foreign"
or "endogenous." If the decision is "foreign," then nothing
stands in the way of a further fight against the antigen.
The recognition of the foreign protein in contact with
endogenous MHC causes further differentiation of the T
lymphocytes to plasma cells, which ultimately secrete
interleukins. This interleukin secretion leads to
activation of B lymphocytes, which, on their part, did
recognize the soluble antigen, but first require the
"communication" from the T lymphocytes via the interleukins
for their own differentiation. This is followed by
differentiation of the B lymphocytes to plasma cells, where,
in atopic patients, relatively large amounts of antibodies
of the IgE class will be secreted.
If the antigen was recognized on the T cell level as
endogenous, then, under normal conditions, the immune
response will be broken off at this point. However, the
complex regulatory cascades of the immune system har~or a
number of possible defects. Witnesses of this condition are
the many, mostly fatal, clinical cases of autoimmune
diseases, in which the endogenous immune system cannot
differentiate between "self" and "non-self."
Up to now the diagnosis and thus the therapy of allergic
diseases have not been satisfactory. Molecular
characterization of the principal allergens of Alternaria by
cDNA cloning, sequencing, sequence comparison of the
allergenic protein with protein databanks and production of
recombinant allergens will give more information on the in
vivo function of proteins which elicit false immune
reactions. This information is of interest for the
following reasons:
21 70355
.
1) Highly pure recombinant allergens can ~e used for a more
careful diagnosis, one that is better than can now be made
with crude extracts.
2) At the same time the sequence of the allergens will help
to define tolerogenic peptides, and possibly also to learn
to understand the IgE class switch which takes place during
immunization with the allergen.
For decades IgE-caused allergies, thus e.g. also allergies
to fungal spores, have been treated by hyposensitization
(Bousquet et al., 1991). This therapy consists in
administering allergen extracts in the form of injections or
by oral application in aqueous form as drops in increasing
doses, until a maintenance dose has been attained over
several years. The result of this treatment is the
achievement of tolerance to the allergens introduced, which
manifests itself in a decrease of disease symptoms (Birkner
et al., 1990). The problem with this type of treatment is
the many adverse reactions which it engenders. In the
course of hyposensitization therapy cases of anaphylactic
shock have occurred during treatment. The problem here is
the difficulty of standardizing the fungal protein isolates.
By using peptides derived from allergens but devoid of
anaphylactic effect it might be possible to administer
higher doses without risk, whereby a substantial improvement
of hyposensitization could be achieved.
Alternaria alternata can be found practically everywhere in
nature. Favored habitats of the fungus, however, are
various soil types, grain silos, rotted wood, but also
living plants, compost sites and bird's nests. When
tomatoes are covered with black spots, they most likely
originate from Alternaria. However, it is not only in
nature that Alternaria alternata may be found. Very often
21 70355
,
the fungus is encountered in humid indoor areas and on
window frames. In general, warm temperatures and high
atmospheric humidity favor the growth of the fungus.
At present Alternaria alternata is considered one of the
most important allergy-inducing fungi. Yunginger et al.
(1989) have characterized the first allergic fraction, and
isolated Altal, the first protein acting as allergen. A
great problem with Alternaria alternata is the wide range of
variation of the fungus: Variations in protein pattern and
a variable potency of the triggering of allergy have
frequently been described. Nyholm et al. (1983) showed that
Agl and Alt-1 are the same allergen, but that different
strains of Alternaria alternata may show a range of
variation of the protein. The review article of Budd (1986)
describes the isolation of the allergic protein Alt-1, now
Altal. So far, complete cDNA sequences of allergic proteins
of Alternaria alternata have not been published. However,
various studies show strong cross reactions between
Alternaria alternata, Stemphylium and Curvularia (Agarwal
1982).
Created in accordance with the invention are recombinant DNA
molecules of the type mentioned in the introduction, which
have nucleic acid sequences that correspond in homologous
fashion to sequences 1, 3-5, 7-9 as well as 12 and 13, or to
partial regions of these sequences, or have nucleic acid
sequences which hybridize with the aforementioned sequences
under stringent conditions. The DNA molecules can also have
nucleic acid sequences that can be derived from the
aforementioned sequences by degeneration.
Further features of the subject matter of the invention will
become evident from the following presentation.
21 70355
a) Description of the allergic proteins of Alternaria
alternata by Western blotting
For cloning the present allergens of Alternaria alternata,
sera of 142 patients were available. To test the reactivity
of the patients with fungus protein extract, Alternaria
alternata (collection of Prof. Windisch, Berlin, No. 08-
0203) was cultivated on solid medium (2% glucose, 2%
peptone, 1% yeast extract). For the protein extraction the
fungal mat was lifted after 3 days of growth at 28C and
broken up with liquid nitrogen. Separation of the extracted
proteins was carried out on a denaturing polyacrylamide gel,
which was subsequently blotted, incubated with patient serum
and detected with l25I-labeled anti-human IgE. Expressed in
percentages the patients reacted to the allergenic proteins
as follows:
Alta53 44.8%
Alta22 3.4%
Altall 10.3%
When protein was isolated from fungus material purchased
from Allergon (Sweden) and used for the immunoblot, almost
the same band pattern was detected. As can be seen from
these numbers, Alta53 is a principal allergen and Alta22 and
Altall are secondary allergens.
Fig. 1 gives an overview of the patient material available
for cloning the above-described allergens. The figure shows
a 12.5% polyacrylamide gel. Patients No. 35 and 40 (these
are also the patients who were used for the subsequent
screening) show bands of the order of 53 kD, 22 kD and
11 kD.
2 1 70355
.
Thus Fig. 1 shows a Western blot of a 12.5% polyacrylamide
gel after separation of Alternaria alternata protein
extract; incubation with sera of different patients;
detection with l2sI-labeled anti-human IgE.
b) Construction of the cDNA expression bank
Total RNA was obtained by the acid guanidium-phenol
extraction method from fungus material cultivated by us.
Poly(A)plus enrichment was done with oligo(dT) cellulose
obtained from Bohringer. The cDNA synthesis (first and
second strand) was performed as described in the "Manual des
Lambda ZAP-Systems" [Manual of the lambda ZAP system] of
Stratagene Co. The cDNA was then provided with EcoRI (on
the 3' side) and XbaI linkers (on the 5' side), ligated in
predigested lambda-ZAP arms, and packaged. The titer of the
primary bank was 900,000 clones.
c) Screening of the cDNA bank with patient sera, in vivo
.
exclslon, sequenclng
The expression bank was screened by incubation of the
"lifted phage plaques with a serum mixture of 2 patients,
for which it was known, from Western blotting, that they
cover the spectrum of the detected antigens. Detection was
again done with anti-human IgE RAST antibodies of Pharmacia
Co. After secondary and tertiary screening 150 positive
clones remained. From 12 clones the already readily
sequenceable Bluescript vector was excised in vivo with the
aid of a helper phage (procedure as in the manual of the
lambda ZAP kit). Restriction digests of the excised
plasmids (EcoRI-XbaI double digests) showed 3 different
insert types. These 3 clones were sequenced according to
Sanger~s method (Sanger, 1977).
21 70355
d) Expression of the Alta53, Alta22 and Altall cDNAs as
~-galactosidase fusion protein
By means of the above-described IgE screenings 3 complete
cDNA cones were obtained. The respective recombinant
plasmids were transformed into the E. coli XLI-Blue strain
and induced with IPTG (isopropyl ~-D-thiogalactopyranoside).
The E. coli total protein extract was then electro-
phoretically separated and blotted on nitrocellulose. The
fusion protein was detected with serum IgE of patients
allergic to fungus and with an 125I-labeled rabbit anti-human
IgE antibody (Pharmacia, Uppsala, Sweden).
The next 2 figures show the recombinant ~-galactosidase
fusion proteins after incubation with patient serum and
detection with iodine-labeled anti-human IgE. The ~-
galactosidase part of the fusion protein contains 36 amino
acids, which corresponds to a molecular weight of 3800
daltons. The following Figs. 3 and 4, too, should be viewed
by taking this "enlargement" of the allergenic protein into
consideration. Lanes (clones) 1, 2, 4, 5, 6, 7 show the
recombinant fusion protein Altall, lanes 3 and 12 the
recombinant fusion protein Alta22, and lanes 8 and 10 the
recombinant Alta53 which has been enlarged by the fusion
portion.
Figs. 2 and 3 thus show an expression of the recombinant
proteins Alta52 [sic], Alta22 and Altall in Bluescript after
IPTG induction.
e) Determination of B- and T-cell epitopes in the
recombinant allergens
The derived amino acid sequence of the allergens offers the
prerequisite for the prediction of B- and T-cell epitopes by
21 70355
.
means of appropriate computer programs. Through these
studies it is possible to define specific T- and B-cell
epitopes which have the capacity e.g. of stimulating T
lymphocytes and inducing them to proliferate, but also (in
the case of an exactly defined dose) of bringing the cells
into a state of tolerance or nonreactivity (anergy)
(Rothbard et al., 1991). Each of the epitopes determined
will be cited in the description of the recombinant protein
in individual figures.
The search for B-cell epitopes was carried out with the aid
of the GCG program (Genetics Computer Group) "PROTCALC",
which, however, was extended by the addition of important
parameters by Prof. Modrow's study group. The determination
is based on weighing the parameters hydrophilicity (Kyte-
Doolittle), secondary structure (Chou-Fasman), surface
localization (Robson-Garnier) and flexibility, whereby the
antigenicity of partial peptides is calculated.
The principle of T-cell epitope prediction was carried out
essentially according to the algorithm of Margalit et al.
(1987). The principle consists in looking for amphipathic
helices according to primary sequence of the peptide to be
determined, flanked by hydrophilic regions. For relevant T-
cell epitopes the calculated score must be greater than 10.
In the case of the MHC II-associated peptides no consensus
can be defined on the basis of either the sequence or the
length of the peptide, as in the case of HLA-A2 (human
leukocyte antigen) (MHC I)-associated peptides. In the case
of HLA-A2-associated peptides the length of the peptide is
10 amino acids, the second amino acid being a tyrosine and
the last amino acid a leucine (Rammensee et al., 1993). The
calculated epitopes will be separately cited in the
description of the individual allergenic sequences.
` 21 70355
Molecular characterization of the cloned fungal allergens
(sequence protocols)
Below, the cDNA sequences and the analyses carried out with
them are presented in succession. Computer evaluation of
the following sequences was done on an Ultrix-DEC 5000 work
station with the aid of the GCG software packet (= Wisconsin
packet: the algorithms of this packet were developed by the
University of Wisconsin).
A. Alta53
The foIlowing Sequence 1 shows the complete cDNA sequence of
Alta53, beginning with the initiating ATG. The length of
the cDNA is 1488 bp, which corresponds to a calculated
molecular weight of 53543 daltons. Thus on the basis of the
molecular weight the observed band in the Western blot at
53 kD correlates with the cloned and sequenced allergen. On
the basis of analysis carried out so far, there is probably
no signal peptide before the mature protein.
Sequence: Alta53=ALDH_alt -> 1-phase translation 53543
daltons
(1) INFORMATION ON SEQ ID NO:1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1488 base pairs / 496 amino acid groups
(B) TYPE: Nucleic acid / protein
(C) STRAND FORM: ds
(D) TOPOLOGY: Linear
(ii) NATURE OF MOLECULE: cDNA to mRNA / protein
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) NATURE OF FRAGMENT: Total sequence
12
21 70355
(vi) ORIGINAL SOURCE:
(A) ORGANISM): Alternaria alternans /sic7
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
DNA sequence 1488 bp ATGACATCTGTA ... CTGTTCGGTTAA linear
~1 / 1 1 -
ATC ACA TC~ C7A AAG cTc 7C~ ACC CCr CA~ ACG CGC C~G SSC GAG CAO CCC Acc CCA C7c
r~e~ th~ Jer v~l ly~ lcu er ehr pro qln ehr gly qlu phe vlU vln pro thr gly leu
61 / 2Z 91 / ~1
STC ATC ~AC AA7 CAG Trc G7A AA~ GCT cr7 CAC CGC AAO ACC 17T GAT C77 A7C AAC c
phe lle ~sn ~sn qlu phe v~l lys ~l~ v~l ~sp gly ly- 'hr phe ~p v~1 lle Isn pro
122 / ~1 lSl / Sl
SCC ACr CAG GAO GIC ASC SCC AGT G7T cAc GAC CCC ACC CAO A~C GAT GSS CAC A GCT
~e~ thr glu glu v~l 11 cy~ ~r v~l gln glu 11~ thr ylu ly- ~rp v~l lsp ~le ~1
1~1 / 61 221 / 71
Csr GC7 GCC CCC CCC AAA GCC SSC AAC CGC CCA SCC qcA AAC CAa ACA CCA CAC AA- AGG
v~l ~1~ ~ rg ly~ ph~ ~n gly pro trp ~1~ ly- glu thr pro qlu ~5A ~V
241 / ~ 271 / 91
GGA AAO CT0 crr AAC AAC c7r CCC CAT CrG SSC GAa AAa AAT CCC CAC CTC A~S CC~ GCT
gly ly~ l~u leu ~n ly~ l~u ~ sp l~u phc ylu ly- ~sn ~1~ ~5p leu lle
301 / lûl 331 / 111
CTC cAa Gcr crC GAC AAC CGC AAa GCC 52C ACC Asa GCC AAC AAC GIC CAI GST CCC GCC
v~l qlu ~ u ~rp ~n gly ly~ pbc ~er ~et ~1~ ly- ~-n v 1 ~sp v~l pro ~1
361 / 122 391 / 131
GCC CC7 GG7 IGC cr~ ~GO IAC 7AC GGA GGA TGC GCC CAC AAC ASS GAa GCC AAC G C GSC
~ yly cy~ l~u ~ry tyr tyr gly qly trp ~ sp ly- lle ylu vly ly- v~ v~l
22 / 1~1 ~Sl / 151
CAC ~CA GC~ CCC CAC AGC SIC AAC SAC ATC CCC AAG ACC C7A STC CSC S7S GCG G7C ~GA
~sp thr ~11 pro ~5p ~er phe ~n tyr lle ~r~7 ly~ ~cr l~u leu v~l phe ~1~ v~l ~ry
4ûl / 16' 511 / 171
SCA SCC ATC CAA CST CCT AS7~ C7~C ASC SCC SCA SCC AAG ASS GGT CCS GCC ATC GCC ACS
~er ~er ret qlu leu pro lle leu ret trp ~er trp ly~ lle yly pro ~1~ lle ~1~ thr
5-1 / 181 571 / 191
GGS AAC ACC CTC C7`C C7.'C AAG ACS GCS CAG CAC ACA CC7 CSC TCC GCA SAC ASS CCC SGC
vly ~-n thr v~l vll leu ly~ thr ~1~ ylu yln thr prO leu er ~1~ tyr lle ~1~ cy~
601 / 2ûl 631 / 221
AAG CSC ASC CAa GAa GCC GGS SSC CCA CCA CGS GrC ATC AAC GTC ASC A~ST GGS SSC GGA
ly~ leu lle yln glu ~1~ gly phe pro pro yly v~l lle ~n v~l lle thr gly phe gly
661 / 222 691 / 231
AAG ASC CCC GGT GCS GCC ASG TCC GCS CAC ASa CAC ASS GAC AAO AST GCC S7S AC. GGS
ly~ lle ~1~ yly ~1~ ~1~ ~et ~er ~1~ hl~ cet ~sp 11- ~sp ly- lle ~1~ phe thr gly
722 / 2~1 751 / 251
SCA ACC G7S CSC GCC CCS CAA ASC ASC AAo SCS GCC GCr GGC ICC AAC SSC AAO AAG GSC
er thr v~l v~l ely rV Vln 11- r~et ly~ ~er ~ gly ~er ~n leu ly~ ly~ v~l
781 / 261 811 / 271
AC7 cr~T CAO C7C GGA GGC AAG AGC CCC AAC AlS GlC SSC GCC GAC GCA GAS CSS CAC GAC
tbr leu qlu leu vly vly ly~ ~er pro ~n ~le v~l phe ~1~ ~sp ~1~ ~sp leu ~sp glu
8~1 / 281 871 / 291
GC7 ATC CAC TGC CTC AAC S7~ CGS AS~ SAC SSC A~C CAC GGA CAG GCS SGS SCS CCS CGS
~1~ lle hl~ trp v~l ~sn phe vly lle tyr ph~ ~n hl~ gly gln ~11 cy~ cy~ gly
901 / ~01 931 / 311
SCG CCS ATC SAC CSC CAA CAA GAO ASC SAC CAC AAa SSC ASC CAC CGC S rc AAC G~0 CCC
er ~rV lle tyr v~l gln VlY glu lle tyr ~sp ly~ pb~ lle gln ~rg phe ly~ qlu ~ry
961 / ~22 991 / 331
GCS CCS CAC AAC GCS GlS CCS CAC CCA SSC GCC CCC ACA CSC CAC GCS CCS CAA CSC SCG
~ yln ~n ~1~ V~l gly ~sp pro ph~ rl~ tbr leu vln gly pro gln v~l ~er
1022 / ~1 lOSl / ~51
c~a CTC CAC SSC GAC CCS ASC ATC GGC SAC ATC cAa GAa GGC AAa AAa T~S CCC CcC ACC
vln leu vln ph- ~5p rv lle ~et vly tyr lle vlu vlU vly ly- ly- er vly ~1~ thr
~081 / ~61 1111 / ~71
ASC c~a ACS CGS GCC AAC CCT AAC CCT CAC AAo CCS SAC STC ASC GAC CCc ACA ASC SSC
~le VlU thr vly gly ~n ~rv ly- gly ~sp ly- gly tyr pbe lle Vlu pro thr lle phe
11~1 / ~81 1171 / ~91
TCC AAC CTA ACC CAa CAC ATG AAo ASS CAC CAA C~A GAa ASC S7`C GGC CCC GSC SCC A~A
~er ~n vll thr vlù ~sp ~et ly~ lle vln vln ql~ vlu lle pb~ vly pro v~l cy~ thr
1201 / ûl 12~1 / ~11
ASC SCC AAC SSC AAa ACA AAa CCC CAC GSC ASC A~o ASS CGC AAC AAC ACC ACA SAC GGS
11- er ly~ ph~ ly~ thr ly~ sp v~l lle ly~ vly ~n ~n thr tbr tyr vly
1261 / ~22 12Jl / ~1
Ctt tcC CCC CCS GSA CAC ACA SCC AAC CTC ACC ACS GCC ASC GAA GS7 CCC AAC CCG CSC
leu ~er ~ v~l bl~ thr ~er ~n leu thr thr ~1~ lle glu v~l ~1~ ~n ~1~ leu
1~22 / ~ 51 / ~Sl
CL7S GCA CGA ACS CSC SCa CTC AAC SCC SAC AAC ACS CSS CAC SGC CAL7 CTS CCC SSC G~A
~rg ~1~ vly tbr v~l trp v~ n er tyr ~n thr leu bl- trp vln leu pro ph~ gly
1381 / ~61 1~ 71
GGG SAC A~C CAO SC7 CG7 ASS GGG CCC CAC SSG GCA cAa GCC CCG crc CAC AAC SAC ASC
vly tyr ly- ylu ~ yly lle yly ~rq qlu l~u 51y ylu ~ leu ~sp ~sn tyr le
~ 1 " " / '9'
CAO ACC AAC ACC Gsa scr AS7 CGS CSS CGC CAS GTS CSC SSC CGS SAA
yln thr ly~ thr v~l ~r lle ~rq leu vly ~sp v~l l~u phe 91y GC~
13
21 70355
Searches for homology with Alta53 in the SWISSPROT protein
databank showed that Alta53, like Clah53, is an aldehyde
dehydrogenase. As proof of the high homology (identities
exist over many stretches) the following Sequence 2 shows a
pairwise alignment between Alta 53 and Clah53. The identity
(identical amino acids) of the two proteins (allergens) with
one another is 78%. The degree of homology (identities plus
homologous amino acid exchanges) is even as high as 86%.
Sequence 2: ALDH
1 MTSV~La~QI~r QPI~L~lNN~v~AVD~uvlN~5~ tVICSVQ 50
1111.1.11:.1.:11111111111111: :1111111111.1.11. 1:
1 MTSVQLETPESG~YEQPTGL~lNN~r v~Gy~ G~L~VVINPSDESVITQVS 50
51 EATERDyDIAvAA-A~uu-FNGpuARETpr~RrTN~AADLFE~NA-vLIAA 100
111111111111111.11:1.1 11111111111.11:11111.11:11
51 EATE~DVDIAVAAARQAFEGsWRLETPFNR~T~TTN~TTANTT`EgNTDLLAA 100
101 vr!ATnr~AT~sMAgNvDvpAAAGcLRyyGG~ADgIEG~vvDTApDsFNyI 150
11.111111 111: 1. :.1.1111111111111.111:11.11.111:
101 vF~T~NrT~ATsMAR.vTcArA~GrrrlyyGGwA~v~ N~v 149
151 Rg.SLLVFAvRssMELPILMwsw~IGpAIATGN~vvL~TAEQTpLsAyIA 199
:1 .: I 1:111:111.11111111-11111111111111::.:1
150 ~;EpIGvcKsD~sLELpLL~wAw~ pATAr~:~vvr~TAEQTpLGGLvA 199
200 cgLIQEA~vlNvl~ ~fiAAMcArnMnID~IA~;~s~v~KQIM 249
..1:.111111111111.1111:1111:1.111:11:1111111111 1:
200 AsLvREA~vlNv~sGFG~vA~AAT~sRMnvD~vAFTGsTvvGRTIL 249
250 TC~r-C~TT~V~TF-~G~T<~P~NIVF~nAnTnFA~HWVN~ N~GQACCA 299
1.11:1111111111111111111.111:1:11 111111:11111.111
250 T~ACS~T~VTTF-T,GGT~NlV~ uADIDh-AI~wvN~ N~GQcccA 299
300 GSRl~vy~;~ lQRF~ERAAQNAVGDPFAA.TLQGPQVSQLQFDRrM 348
111:1111.11111:1:11111..1.1111111 1:111111.:111111
300 GSKV~VY Sl~u~vy~KAQ~NVVGDPFAADTFQGPQVS~VQFDRIM 349
349 ~ K~A .l~G~k~ r~I~lL~ ~Nv~ru~IQQ~ ~V 398
- :11:.11.. 111:1111.11111111111111111111111 .1111111
350 EyIQ~rnA~A~v~G~K~GDR~ ~Nv~ UMRIV~EEIFGPV 399
399 CTIs~F~TRADvI~I~N~;~sAAy~TsNLTTAIEvANAL-KAGTv~vN 448
1.1.11111.1.11:11..1111.11111.11.11111.111:1111111
400 CSIA~E~TRED~TTcT~NA~TyGLAAAv~TRNLNTArEvsNAL~AGTv~vN 449
449 SYN~L~WQLPFGGYIC~ESGIGRELGEAALDNYIQT~TVSIP~LGDVLFGZ 496
.11~11 1:1111111111111111.11.11.11111111111.1111
450 TyNTL-R-Qh~G~sGIGpFTfiFn~T~N~lQ~ sIp~LGDALFGz 497
14
21 70355
The NAD-dependent ALDH is the main enzyme involved in man in
the oxidation of acetaldehyde, a primary product of alcohol
metabolism. In this connection, isoenzymes can often be
found (Harada et al., 1982). Found in man, for example, is
the isoenzyme ALDH I in mitochrondria, and ALDH II in
cytoplasm. Interestingly, in Asian individuals the absence
of ALDH I is no rarity tHarada et al., 1982). The ALDH I
deficiency results in a high acetaldehyde level, which
manifests itself in the form of the so-called flushing
syndrome and other vasomotor symptoms after alcohol
consumption. The isoenzyme loss may be attributed to a
mutation which changes the structure of the native protein
(Hsu et al., 1987). At present the connection between ALDH
and the triggering of allergy is not yet known.
The following Sequence 3 shows the regions of high antigenic
index identified by computer search. These regions
represent highly potent B-cell epitopes.
Sequence 3: Alta53=ALDH_alt: B-cell epitopes
(1) INFORMATION ON SEQ ID NO:3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
21 70355
Val Lys Leu Ser Thr Pro Gln Thr Gly Glu Phe Glu Gln Pro Thr Gly
(4-l9)
Ala Val Asp Gly Ly~ Thr Phe (29-35)
Ile Asn Pro Ser Thr Glu Glu (38-44)
Gly Pro Trp Ala Lyg Glu Thr Pro Glu Asn Arg Gly Lys Leu Leu Asn
(70-85)
Leu Arg Tyr Tyr Gly Gly Trp Ala Asp Lys Ile Glu Gly (125-137)
A~p Thr Ala Pro Asp Ser Phe Aqn Tyr Ile Arg Lys Ser (141-153)
Glu ~la Gly Phe Pro Pro Gly Val (205-212)
Gly Ser Asn Leu Lys Lys Val Thr Leu (254-262)
Glu Leu Gly Gly Lys Ser Pro Asn Ile (263-271)
Tyr Ile Glu Glu Gly Lys Ly~ Ser Gly Ala Thr (350-360)
Ile Glu Thr Gly Gly Asn Arg Ly~ Gly Asp Ly~ Gly Tyr Phe Ile Glu
(361-376)
Ile Gly Asn Asn Thr Thr Tyr Gly (413-420)
Ala Val ~is Thr Ser Asn Leu Thr (424-431)
The following Sequence 4 shows the amphipathic helices
determined with the aid of the computer program, and which
are flanked ~y hydrophilic regions. Such regions, having a
score of more than 10, represent possible T-cell epitopes.
21 70355
Sequence 4: Predicted amphipathic segments
T-cell epitopes
_______________
EFEQ
FVKAVD
~CI FDVI
KAFNGPWA
KLLNKLADLFE
IAAVEALDNGKA
MAKNVDVP
AAGCLRYYGGWADKIEGK
VDTAPDSFNY
GVINVITGFGKI
IYDKFIQRFKERAA
~AVGDPFAAT
QFDRIMGYI
GPVCrI
AIEVANALR
RELGEAALD
(1) INFORMATION ON SEQ ID NO:4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
21 70355
.
Glu Phe Glu Gl~ ~ 16)
Phe Val Lys Ala Val Asp (26-31)
Ly~ Thr Phe Asp Val Ile (33-38)
Lys Ala Phe A~n Gly Pro Trp Ala (66-73)
Ly~ Le~ Leu Asn Lys Leu Ala Asp Leu Phe Glu (82-92)
Ile Ala Ala Val Glu Ala Leu Asp Asn Gly Lys Ala ~98-109)
Met Ala Ly~ Asn Val Asp Val Pro (112-119)
~la Al~ Gly Cys Leu Arg Tyr Tyr Gly Gly Trp Ala Asp Lys Ile Glu Gly
Lys (121-138)
Val Asp Thr Ala Pro Asp Ser Phe Asn ~yr ~140-149)
Gly Val Ile Asn Val Ile Thr Gly Phe Gly Lys Ile ~211-222)
Ile Tyr Asp Lys Phe Ile Gln Arg Phe Ly~ Glu Arg Ala Ala (309-322)
A~n Ala Val Gly Asp Pro Phe Ala Ala Ihr (324-333)
Gln Phe Asp Arg Ile Met Gly Tyr Ile (343-351)
Gly Pro Val Cys Thr Ile (396-401)
Ala Ile Glu Val Ala Asn Ala Leu Arg (433-441)
Arg Glu Leu Gly Glu Ala Ala Leu Asp (469-477)
The T-cell epitopes are calculated from the amino acid
positions of the midpoints, which are flanked N-terminally
by a lysine (K) and C-terminally by a p~oline (P) (- flags).
Potential T-cell epitopes are present only when the score
index is greater than 10.
B. Altall
The following Sequence 5 shows the complete cDNA sequence of
Altall and of the amino acid sequence derived therefrom.
The open reading frame comprises 342 bp or 114 amino acids.
18
21 70355
The calculated molecular weight is 11127 daltons and thus
corresponds to the 11 kD antigenic protein, which is
recognized in the Western blot by 10.3% of the patients.
Sequence 5: Altall=rla2_alt -> 1-phase translation 11127
daltons
(1) INFORMATION ON SEQ ID NO:5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs / 114 amino acid groups
(B) TYPE: Nucleic acid / protein
(C) STRAND FORM: ds
(D) TOPOLOGY: Linear
(ii) NATURE OF MOLECULE: cDNA to mRNA / protein
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) NATURE OF FRAGMENT: Total sequence
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
DNSA sequence 342 bp ATGAAGCACCTC ... CTCTTCGACTAA linear
1 / 1 31 / 11
A~o AA~ C~C CSC GCa GCA SAC CTC CTC CTC GGC cr~ GG7.~ GGC AAC ACc ~ W CCC ~CC GCT
~et lys h~s leu ~1~ tyr leu leu leu gly leu gly gly ~sn thr .er pro ~er ~161 / 22 91 / 31
GCC GAC GTC AAa GCC GTC C~ G~a TCC GTT GCT ATC GAa GCT GAC TCC G~C CGS C5~ C~C
~ sp v~l ly~ v~l leu glu ser v~l gly ~le glu ~ sp ser ~sp ~rg leu ~sp
122 / ~1 lSl / Sl
AAa Csa ATC ~CC GAC C7.~ CA0 GGC ~Aa GAC ATC ~AC GAa CSC ASC GC~ SCC GG~ ~CC G~Gly- leu lle .er glu leu glu gly lys ~sp ~ln ~sn glu leu ~ .er gly er glu
181 / 61 221 / 71
AA0 C~T GCT TCC Gl~ CCC SCC GGT GGT GCC Gq~ GGT GCT GCC CCT SCC GsT GGT GcT GC~rly- leu ~1~ ser v~l pro ser gly gly ~1~ gly gly ~ er gly gly
241 / ~1 271 / 91
GCC CCT GGT GGC TCC GcT cAa GCT GAC GcC GC~ CCl GAG GCc GCC AA3 G~D GAG GA3 A~G11~ ~1~ gly gly ser ~1~ gln ~1~ glu ~1~ pro glu ~ ly~ qlu glu qlu iy-
301 / 101 331 / 111 .
GAa GAa ~CT GAC GAD cAC ATa GGT TTC GCT CTC ~SC CAC IAA
glu glu ser ~sp glu ~sp ~et gly phe gly leu phe ~sp OCH
19
21 70355
.
In this case, homology searches in the SWISSPTOT protein
databank showed homologies to the ribosomal protein P2.
This ribosomal protein is involved in the formation of the
large subunit of the ribosomes. Thus a homology to Clahll,
the counterpart of Altall in Cladosporium herbarum, is also
necessarily present. The identities and homologies between
Altall and Clahll can be seen from the following Sequence 6.
The identity of the two proteins is 74%, the degree of
homology increases to 84%, which undoubtedly points to a
similar function of these two proteins.
Sequence 6
rla2_alt x rla2_clado
1 MR~LAAYL~LGLGGNTSPSAADVRAVIESVGIEADSD~LDRLISELEG~D 50
11.111:11111 11.1111.1:1.11.1111:11.:. : 1:.111111
1 M~yLAAF~LLGL.GNsspsAEDIRrvLssvGIDADEEpsQ~TTRF-TF-GRD 49
51 INELIA5GSERLASVPSGGAGGAAA5GGAAAAGGSAQAEAAPFA~EEE~ 100
11111.111111111111111:1.1:1:111:1 1.1 1.11.1
50 INELISSGSERLASVPSGGAGAASAGGAAAAGG....... AEERAEEERR 92
101 EESDED~GFGLFDZ 114
l l l l : l l l l l l l l l
93 EESDDDMGFGLFDZ 106
Acidic ribosomal proteins (such as PO, Pl and P2) of
different organisms have been analyzed by many techniques.
A distinction is made between A proteins (acidic) and P
proteins (phosphorylated A proteins). One feature of A
proteins is the large number of hydrophobic amino acids.
For that reason they can readily be dissociated from the
ribosome (50% ethanol and high salt concentration). The
best-characterized A protein in prokaryons is the L7/L12
protein of Escherichia coli. The eukaryotic homologues are
the proteins Pl and P2, which, like the L7/L12 protein,
interacts with the elongation factQr EFl and EF2.
21 70355
The C-terminal sequence contains an epitope that is
recognized by autoantibodies of lupus patients (Francoeur et
al., 1985; Rich et al., 1987; Hines et al., 1991). In its
homology the P2 protein corresponds to the allergenic
protein Altall.
The B-cell epitopes shown in the next Sequence 7 were
calculated by taking the secondary structure, surface
position, hydrophilicity, flexibility, etc. into
consideration.
Sequence 7: Altall=rla2_alt: B-cell epitopes
(I) INFORMATION ON SEQ ID NO:7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Leu aly Gly ~sn ~rhr Ser Pro Scr Al~ ~sp ~12-22)
Glu ~1~ Asp Ser ~sp Arq Leu ~sp Ly~ Leu Ilc 8er (32-44)
Glu Leu Olu Gly Ly~ A5p Ile A~n Glu Leu (4S-54)
8er Gly Ser Glu Ly~ Leu .~1~ Ser (S6-64)
Pro Glu Al~ Al~ Ly~ Glu Glu Glu Ly~ Glu Glu Scr Asp Glu A~p ~et Gly Phe (92 - 109)
The following Sequence 8 shows the calculated T-cell
epitopes and represents the amino acids in the 1-letter
code.
21
21 70355
.
Sequence 8: Predicted amphipathic segments
T-cell epitopes
_______________
RLDKLISELEGKDINEUASG
EKLASVPSGG
(1) INFORMATION ON SEQ ID NO:8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Ar~ Leu Asp Ly~ Leu lle Ser Glu Leu Glu Gly Ly~ Asp Il- Asn Glu Leu Ilc Al~ Ser Gly
~3~-S~ ~
Glu Ly~ Leu All Ser V~l Pro Ser Gly Gly (60-69)
The T-cell epitopes are calculated from the amino-acid
positions of the midpoints, which are flanked (= flags) N-
terminally by a lysine (K) and C-terminally by a proline
(P). Potential T-cell epitopes are present only when the
score index is greater than 10.
C. Alta22
The following Sequence 9 shows the complete cDNA sequence of
Alta22. The primary sequence derived therefrom can also be
seen from the sequence. The open reading frame of the
allergenic protein is 615 bp, which corresponds to an amino
22
21 70355
acid length of 205 amino acids. The calculated molecular
weight of the recombinant protein is 22041 daltons. On the
basis of analysis carried out so far no signal sequence
stands in front of the mature protein.
Sequence 9: YCP4_alt -> l-phase translation 22041 daltons
(1) INFORMATION ON SEQ ID NO:9
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 base pairs / 205 amino acid groups
(B) TYPE Nucleic acid/protein
(C) STRAND FORM: ds
(D) TOPOLOGY: Linear
(ii) NATURE OF MOLECULE: cDNa to mRNA / protein
(iii) HYPOTHETICAL: No
(iv) ANTISENSE: No
(v) NATURE OF FRAGMENT: Total sequence
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
DNA sequence 615 bp ATGGCTCCCAAG ... GCGCATCAGTGA linear
1 / 1 31 / 11
AS~ GC~ CCC AAa ASC GCC ATS GSG IAC SAC SCC ATG SAC G~C CAC ASC AA¢ AAa Ata GCC
~et ~1~ pro ly~ lle ~1~ lle v~l tyr tyr ~er ~et tyr gly hl~ ~le ly~ ly- ~et ~1
61 / 22 91 / 31
G~S GC~ GAG S7G AAG GGS ATC CAA GA¢ GCS GGC GGS GAS GCC AAa CSC SSC CAA GSC GCC
~p ~1~ glu leu ly~ gly lln gln qlu ~1~ gly gly ~sp ~1~ ly~ lcu phe gln v
122 / ~1 151 / Sl
CAa ACC c~a CCS CAO GAA GSC CSC GAC AAa ATa SAC Gca CCC CCC AAS GAC SCA sca GSC
~lu thr leu pro gln glu v~l leu ~sp ly~ ~et tyr ~1~ pro pro lyJ ~p ~er ~er v~l
181 / 61 221 / ~1
CCC GSC C~C CAG GAC CCA GCC GIC CSC GAA GAA S7.~ GAC GCC ASC C7~C SSC GGC AIC CCC
pro v~l leu glu ~5p pro ~11 v~l leu glu glu phe ~sp gly lle leu phe gly lle pro
2~1 / 81 271 / 91
ACC CGC SAC GGC AAC SSC CCC GCA CAA SSC AA¢ ACC S7C SGa GAC AAG ACA GGC AAa CAA
t~r ~rg tyr gly ~n phe pro ~1~ gln phe ly~ thr phe trp ~sp ly~ thr gly ly~ gln
301 / 101 331 / 111
SG~ CA~ CAA G~C GCC SSS SGG GGA AA¢ SAC GCC GGS C7C SSC GS~ SCC ACG G~C ACC CSG
trp gln gln gly ~1~ phe t}p gly ly~ tyr ~1~ gly vll phe v~l ~er thr gly thr leu
21 70355
.
36~ / 122 391 / 13~
aaCGGGT GGC CAG GAG ACG ACT GCC ATT ACC AGC ATG AGC ACG CrT CTC GAC CAC GGT rTC
~ly gly gly gln glu thr thr ll~ llc t~r ~er ~et er thr leu v~l ~sp ~l~ qly phe
~22 t 141 451 / 151
ASC SAC G~T CCC C~T GGC TAC AAG ACT Gca m ACC AIG TTa GCC AAC TTa GAC CAa GTC
Llo tyr vll pro leu qly tyr ly5 thr ~1~ phe ~er ~rt leu ~1~ ~sn leu Isp glu v~l
481 / 161 Sll / 171
CAC GGT GCA AGC CCA TGa GGT GCT GGT ACC TIC SCT GCC GGC GAT GGA Tca AGa CA~ CCC
hl- gly gly ~er pro ~rp gly ~11 gly thr p~Q ~er 11~ gly ~p gly ~er ~tg ~ln pro
S~l / 181 571 / 191
AGr CAa C~T GAG CTC AAC AIT GCG CAG GCT CA5 GGT A~G GCT TTC TAC GAG GC~ C~T GCC
~er glu leu glu l~u ~sn Lle ~1~ gln 1l~ qln gly ly~ phe tyr glu ~1~ v~l ~1
601 / 201
~Aa cca CAT CAa SGA
ly~ hL~ gln OPA
Homology searches with the sequenced protein in the
SWISSPROT protein databank showed that the allergen Alta22
has significant homology with the yeast protein YCP4. The
identity of the two proteins is 56%, and the homology even
increases to 72~. Such high similarity probably permits the
assumption that these two proteins have a common function.
The following Sequence l0 reflects the high homology of
Alta22 and YCP4.
Sequence l0:
ycp4_al1:x ycp4_yeast
1 KAPKIAIvYYsMYGHIKKMADALLxGIQ~AGGDAKLFQvA~TLpQEvLDK S0
1 KV.KIAlITYSTYGHIDVLAQAVXXG~GG~ADl~v~PD~VLTX ~9
Sl MKAPPXDSSVPVLLDPAVI~ZFDGILF~IP~RYG~F,PAa~ ffv~,~Q 100
1 11-1...:11 .:..:11 :1::111:111:11:111:-.111111
SO K~APQKP~DIPVATLXTLLE.YDAFL~vYlnFC~LPAQUSAFUDXTGGL 98
.
101 UQQGA~GXYAc,v~vSI~"LCGGQZTTA~TSKSTLVDHGFIYVPLGY~A 150
1..1.: 11 11:1111: 11111.1- .::1 1..11:1::11111 .
99 UAKGSL~CXAAGIFVSTSSYGCGOZsTvxALLsyLA~GIIFLpLGyK~s 148
151 FSKL~LDEVHGGSPUGAGTESAG~GS~4PS~LEL~LAQAQGXA~YZ.A. 198
1. 11.::111111111111:.:.1111 :1.111.11: 111.111 1
149 FAELAsIEEVHGGSPuGAGTLAGPDGsRTAsPLLLPlALIQGXTFY~TAx 198
199 ... VAK.... ...... .. .A~QZ 205
.1 1 1..:
199 XLFPAX~A~S~LA~lllSDAAKR4 223
24
21 7a35s
Now, what about the function of YCP4? In the Yeast Genome
Project the sequence or the open reading frame of YCP4 was
localized at chromosome 3 of Saccharomyces cerevisiae, and
published (Biteau et al., 1992). According to Biteau et al.
(1992) a disruption of YCP4 showed no phenotype. However,
refined phenotype analyses have indicated that the yeast
YCP4 could have a function as a heat shock protein.
Incidentally, this study shows how important Saccharomyces
cerevisiae can be for the function analysis of allergens.
The ease of transformability, combined with matured methods
of molecular genetics, make it possible to disrupt genes in
yeasts and analyze the phenotype resulting therefrom.
It was also found that Alta22, too, has its homologous
partner in Cladosporium herbarum. The following Sequence 11
shows a multiple sequence alignment between yeast YCP4 and
the allergens Alta22 and Clah22.
Sequence 11:
-- so
p~leup.msf~rcP~ ~ltpro) hAPX~ArVYY SmrC~I~LKA D~lXCIqLA GGCA~LFq~ rrLPQrVLdX
p~l-up-msftscP~ cl~dopro) KAPrIAIlFr ST~CLVqtL~ ~ ~n-r-~ W rv~vLrRVp FrLtQrVLlX
p~leup.msf~ycp~-ye~st) .~vX~A~It~ Sr~GUdVLA q~vLXCVe&A W ~ADlr~Va rrLPd~VLTX
Consen~u~ ---X~AI--r S--CR----A -A--XC---~ CO------V- rrL--rVL-X
51 100
plleup.~sf(YCP~ ~ltpro) ~y~PPXD-SV PVleDP~VI~ eFDg~LFCIP SRYCNFPAQF ~l~nvA-~Q
p~leup.ms~(~cP~ cl~dopro) ffhAPPXDDS~ Pe~TDPfILF qYDrFphChP 5F~G~FPAQ~ rrFUDr~CGQ
p~lcup.muf(ycp~_ye~st) KnAPqKp~ PV~T~tlL~ .YD~FL~CVP T~EG~LPAQ~ ~vX.~l
C~n' - 'U5 ff-AP-X-- - - P-------L --D----C-P ~R-GN-PAQ- --~UD-5C--
101 , 150
plleup.msf(rCP4 1ltpro) UQqGAFRCKr ~v~v~ l GGGQE~L~P~t sffSrLvd~Gf IrVPLCrXT~
p~leup.msf(~rcp~ cl~dopro) RQtCAFRCXY AClFISTCTq CCCCrE:~AlA &ffSrLs8LGI IrVPLGYlCrt
plleup.muf(ycp~ y~st) U~ T-~ry~ ACIFVSS~sy GGGQZSSv~ cLSyL~BGI lFlPLOrXn-
Con~en~u~ U--G----GK- AC-r-Sr--- WGQZ-r--- --S-L--8~- I---PLa~K--
lSl 200
p~leup.~s~(YCP4 ~ltpro) F~nlDZV ~ -Ir FS~GDOSR4P SeLZLnIAqA ~Y~VA
plleup.osf(rcp4 cl~dopro) F~LLLqdns V rC~vUCACr ~SG~va~QP Sq~;ZLelt.A QC;!CAFrr~VA
p~leup.msf IYcp4_ye1~t) F1e~slF V gGaSPUGACr L~OpDCSRt~ SpLZLrIAe~ QC;XtFrl:t~
ConJensus F--L----rV -G---UCACT -----DGSR-- S--l!L----- Q~C-~!---
ZO1 249
plleup.m5f ~YCP4 ~.ltpro~ K~qZ... .......... .......... .......... .........
pllcup.D~sf(rcP4_cl:ldopro) KvnfQz......... ....... ....... -
plleup.ms(y~p4 ye~st} Klfp~ ~p ~te~ttt~d ~:rqt~cp~ ~tt~dO;~d)~ gll~cctvm
Con~n-us K--------- -~---~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ -~~~~~~~~
2 1 70355
The B-cell epitopes found with computer support may be seen
in the next Sequence 12.
Sequence 12: Alta22=YCP4_alt: B-cell epitopes
(1) INFORMATION ON SEQ ID NO:12
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Lyr ~et Syr ~1~ Pro Pro Ly~ Asp Ser Ser V~l (50-60)
Ilo Pro Shr ~r~ Syr Gly Asn Pho Pro (79-B7)
Gln Phe Ly~ Shr Phe Srp A5p Ly~ Shr Gly Ly~ aln Srp Gln Gln Gly Al~ Pho Srp Cly Ly- ~yr
Al~ Gly (89-112)
Oly Ihr Leu Gly Gly Gly Gln Glu Shr Shr A1~ Ilo Shr 8er (118-131)
Leu ~p Glu V~ Gly aly ber Pro Srp Gly ~1~ Gly Shr (lS7-170)
Phe 8er ~1~ Gly ~sp Gly Ser Art7 Gln Pro Ser Glu Leu Glu Leu (171-185)
The following Sequence 13 shows the calculated T-cell
epitopes. Amphipathic regions having a score of less than
10 are not assumed to be relevant.
21 70355
Sequence 13: Predicted amphipathic segments
T-cell epitopes:
_______________
SMYGHIKKMAD
GIQEA
L~:QVAEI LPQEVLDKMYA
AVLEEFDGI
TRyGNFpAoFKTFwDKTGKQw
TAITSMSTL
FSMLANLDEVHG
QGKAFYEAVA
(1) INFORMATION ON SEQ ID NO:13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(~) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPOTHETICAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternans
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
8er ~et Syr Gly H~ Ly~ Ly- ~et Al~ Asp (11-21)
aly Ile aln Glu ~1l (26-30)
Leu Phe Gln V~l A~ Clu Sbr Leu Pro Gln Clu V~l Leu Asp Ly~ ~et Syr Al~ (36-S3)
Al~ V~l Leu Glu Glu Pbe Asp aly Ile (67-7S)
5~r Arg Syr Gly Asn Pbe Pro All Gln Phe Ly- Sbr Phe Srp Asp Ly~ Shr Gly Ly- Gln Srp
(81-101)
S~r Al~ Ile Shr 8er ~et 8er Sbr Leu (127-13S~
Phe 8er ~et Leu Al~ A~n Leu Asp Glu V~l Bl~ Cly (151-162)
aln Gly Ly~ Al~ Pbe Syr Glu Al~ V~ (191-200)
21 7()355
.
The T-cell epitopes are calculated from the amino acid
positions of the midpoints, which are flanked (= flags) N-
terminally by a lysine (K) and C-ter~; n~l ly by a proline
(P). T-cell epitopes are present only when the score index
is greater than 10.
28
- 21 70355
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