Note: Descriptions are shown in the official language in which they were submitted.
CA 02204362 1997-0~-02
Translation from German --
Ref. # 4-35/hh
_______________________
~ WO 96/14407 PCT/AT95/00212
o ~
o
z~ Recombinant Alternaria Alternata Allerqens
Y ~
~ The invention relates to recombinant DNA molecules that
zl code for polypeptides which possess the antigenicity of the
allergens Alt a 45 and Alt a 12, or for peptides which have at
least one epitope of these allergens.
~'~ In particular, the invention relates to the complete cDNA
~ sequences of Alt a 45 and Alt a 12 of Alternaria alternata, as
O ~ well as the peptide sequences derived from these primary
sequences which, in individuals allergic to fungi, lead 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
R ~ diagnosis but also for in vivo or in vitro induction of
immunotolerance or anergy of T lymphocytes.
p Immunologic mechanisms, which have been established in
the course of evolution for providing protection against
t: antigens in the environment, normally can differentiate
" ~ between self and non-self. However, as is often the case in
complex control mechanisms, the immune system is also subject
to a certain error rate and when it breaks down an attack on
the body's tissue takes place. There are four principal
situations in which the body is attacked by its own immune
system. These situations differ in the origin of the antigens
which trigger the attack and in their mechanism and
manifestation. The response may be triggered by environmental
antigens, by an infectious agent (but also by harmless
substances), by tissue antigens coming from another person and
by antigens of the individual himself. The reaction is termed
CA 02204362 1997-0~-02
.
"allergic."
A principal feature in allergies is that an administered
substance induces increased sensitivity or hypersensitivity,
instead of protection. Some of these substances are toxins,
others are harmless proteins.
Today four types of hypersensivity are distinguished:
Types I-IV. Types I-III are mediated by antibodies, while type
IV is mediated by T lymphocytes.
Type I hypersensitivity is also termed immediate type
hypersensitivity, because its effect appears within hours of
antigen contact. In the initial sensitizing phase of this
mechanism, antigen (allergen) gets into the body, is absorbed
by antigen-presenting cells (APC) and processed. The processed
antigen is then presented together with MHC II on the surface
of APC to T helper cells (Th). The Th produce lymphokines
which help B lymphocytes to differentiate into antibody-
producing plasma cells. B lymphocytes recognize the allergen
by their surface receptors and secrete IgE. They bind to
receptors of mast cells and basophils. However, this initial
binding has no evident effect on the cells.
However, when the allergen comes into the body once
again, difficulties arise. The multivalent allergen binds to
IgE and via other epitopes to additional IgE molecules, so
that bridge formation (cross linking) between the IgE
molecules starts. Aggregation immobilizes the receptors and
induces a signal transduction chain, which ultimately leads to
degranulation of the mast cells. Degranulation leads to the
production of prostaglandins and leukotrienes. The released
substances are chiefly histamine and heparin. Histamine
stimulates smooth muscle cells, vascular endothelial cells and
nerve endings. Heparin exerts an inhibiting effect on
thrombocytes. The allergic response is influenced by the
nervous system in the acute as well as the late phase.
Neurotransmitters interact with the corresponding receptors on
effector cells and activate these either through cAMP or
CA 02204362 1997-0~-02
through cGMP. The target cells of the neurotransmitters are
again the mast cells, the smooth muscles and epithelial and
secretory cells.
Not all persons who are exposed to the allergen develop
an allergy. Why? The primary cause of allergic states is
believed to be a defect in the immune system.
Neonatal and postnatal serum immunoglobulin levels,
particularly IgA, are very low. Allergic individuals often
have fewer T lymphocytes (CD8+). Many allergic persons had an
elevated IgE level at birth. Their basophils degranulate more
readily. Some also have a defect in suppressor T cells. The
IgE level increases when T cells decrease.
A strong argument against an immunologic defect lies in
that the asthmatic state may also often be triggered without
participation of the allergen. Advocates of this thesis argue
that the primary cause of allergies is physiologic rather than
immunologic. Perhaps the nerve endings in the smooth muscle,
the secretory glands and the blood vessels of target organs
are genetically hyperactive in allergic patients.
The allergy is triggered by characteristic changes after
a second exposure to the same antigen. The quantity of antigen
required for sensitization fluctuates considerably. Too small
a dose produces no response and too great a dose may end in
protection rather than sensitization. A desensitized state may
be produced when a state of shock has been overcome. But the
level of reactive IgE is restored after a few weeks.
The best treatment is avoidance of the allergen. Since
one hundred percent avoidance is hardly achievable, additional
possibilities lie in the use of antihistamines. Currently, so-
called immunotherapy is also being tried. In this approach, an
inability to respond to specific allergens is induced. For
this purpose, the patient is repeatedly i~lln;zed with a
mixture of allergens. One begins with low doses and slowly
increases them until the patient no longer reacts.
Immunotherapy is successful in hay fever, insect bites and
CA 02204362 1997-0~-02
allergic asthma. Treatment seems to induce a form of partial
immunologic tolerance. However, desensitization of the patient
is never absolute.
Up to now, however, neither the diagnosis nor the therapy
of allergic diseases has been satisfactory. Molecular
characterization of the chief allergens of Alternaria by means
of cDNA cloning, sequencing, sequence comparison of the
allergenic protein with protein databases, as well as
production of recombinant allergens, will give more
information about the in vivo function of proteins that
trigger false immune reactions. This information is of
interest for the following reasons:
1) High-purity recombinant allergens can be 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 ;r-lln;zation with the allergen.
For decades IgE-caused allergies, thus for example, also
allergies to fungal spores, have been treated by
hyposensitization (Bousquet et al., 1991). This treatment
consists in administering allergenic extracts in the form of
injections or by or 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 (~irkner 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
CA 02204362 1997-0~-02
risk, whereby a substantial improvement of hyposensitization
could be achieved.
Alternaria alternata can be found practically everywhere
in nature. Preferred sites or habitats of the fungus are
various soil types, grain silos, rotted wood, but also living
plants, compost heaps and bird's nests. When tomatoes are
covered with black spots, they most likely originate from
Alternaria. But it is not only in nature that Alternaria
alternata may be found. Very often 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.
a) Description of the allergenic proteins of Alternaria
alternata by Western blotting
For cloning the present allergens of Alternaria alternata,
sera of 128 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 28~C 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 125I-
labeled antihuman IgE. Expressed in percentages, the patients
reacted to the allergenic proteins as follows:
Alt a 45 47%
Alt a 12 8%
As these numbers reveal, Alt a 45 is a principal allergen and
Alt a 11 is a secondary allergen.
b) Construction of the cDNA expression bank
Total RNA was obtained by the acid guanidium-phenol
CA 02204362 1997-0~-02
extraction method from fungus material cultivated by us.
Poly(A)plus enrichment was done with oligo(dT) cellulose
obtained from Boehringer. The cDNA synthesis (first and second
strand) was performed as described in the "Mânual 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 gene bank with patient sera, in
VlVO excision, 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 antihuman IgE RAST antibodies of Pharmacia Co. After
secondary and tertiary screening 150 positive clones remained.
The two clones described were sequenced according to Sanger's
method (Sanger, 1977).
d) Expression of the Alt a 45 and Alt a 12 cDNAs as B-
galactosidase fusion protein
The respective recombinant plasmids were transformed into
the E. coli strain XLI-Blue strain and induced with IPTG
(isopropyl-B-D-thiogalactopyranoside). The E. coli total
protein extract was then electrophoretically separated and
blotted on nitrocellulose. The fusion protein was detected
with serum IgE of patients allergic to fungus and with an
iodine-labeled rabbit antihuman IgE antibody (Pharmacia,
Uppsala, Sweden).
The ~-galactosidase portion of the fusion protein
contains 36 amino acids, which corresponds to a molecular
-
CA 02204362 1997-0~-02
weight of 3800 daltons. Taking this "enlargement" into
consideration shows precisely that the recombinant fusion
proteins Alt a 45 and Alt a 12 likewise exhibit IgE binding.
e) Determination of B and T-cell epitopes in the recombinant
allergens
The derived amino acid sequence of the allergens provides
the prerequisite for the prediction of B and T-cell epitopes
by means of appropriate computer programs. Through these
studies it is possible to define specific T and B-cell
epitopes which have the capacity, for example, 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 (Genetics Computer Group) program. 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 protein 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 leucocyte
antigen) (MHC I)-associated peptides. In the case of HLA-A2-
CA 02204362 1997-0~-02
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.
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 using the GCG software package (= Wisconsin package:
the algorithms of this package were developed by the
University of Wisconsin).
The DNA molecules according to the invention therefore
have nucleic acid sequences which correspond in homologous
fashion to the following sequences (1-6), or to partial
regions of these sequences, or nucleic acid sequences which
hybridize with the above nucleic acid sequences under
stringent conditions. The degree of stringency is defined by
0.1 x SSC. The degree of homology should be more than 60%.
A. Alt a 45
The following sequence 1 shows the complete cDNA sequence
of Alt a 45 beginning with the initiating ATG. The length of
the cDNA is 1302 bp, which corresponds to a calculated
molecular weight of 45904 daltons. Thus on the basis of the
molecular weight the observed band in the Western blot at 45
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 1: Alt a 45 45904 daltons
(1) INFORMATION ON SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1302 base pairs/434 amino acid groups
CA 02204362 1997-05-02
(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 alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
DHA sequence 1302 b.p. ATGACCAAGCAG ... GACGAGTTGT M linear
1 / 1 31 / 11
ATG ACC AAG CAG GCT CTC CCC GCC GTC TCC GAA GTC ACC M G GAC ACA CTC GAG GAG TTC
~et thr lys gln ala leu pro ala val ser glu val thr lys asp thr leu glu glu phe
61 / Zl 91 / 31
AAG ACC GCC GAC M G GTC GTC CTC GTC GCC TAC TTC GCC GCC GAC GAC M G GCC TCC M C
lys thr ala asp lys val val leu val ala tyr phe ala ala asp asp lys ala ser asn
121 / 41 151 / 51
GAG ACC TTC ACC TCG GTC GCC M C GGT CTC CGT GAC M C TTC CTC TTC GGT GCC ACC M C
glu thr phe thr ser val ala asn gly leu arg asp asn phe leu phe gly ala thr asn
181 / 61 211 / 71
GAC GCT GCT CTG GCC M G GCT GAG GGT GTC AAG CAG CCC GGT CTC GTC TGT ACA AGT CCT
asp ala ala leu ala lys ala glu gly val lys gln pro gly leu val cys thr ser pro
241 / 81 271 / 91
TCG ACG ACG GCA AGG ACG TCT TCA CCG AGA CCT TCG ATG CGG ACG TAT CCG CGA CTT CGC
ser thr thr ala arg thr ser ser pro arg pro ser met arg thr tyr pro arg leu arg
3~1 / 101 331 / 111
AAG GTC GCC TCC ACA CCC CTC ATT GGT GAG G~T GGC CCC GAG ACC TAC GCC GGA TAC ATG
lys val ala ser thr pro leu ile gly glu val gly pro glu thr tyr ala gly tyr met
361 / 121 391 / 131
GCC GCT GGC ATT CCC CTC GCA TAC ATC TTC GCC GAG ACT CCC GAG G M CGT GAG GAG m
ala ala gly ile pro leu ala tyr ile phe ala 9lu thr pro glu glu arg glu glu phe
4Zl / 141 451 / 151
GCC AAG GAG CTG M G CCC CTC GCT CTC M G CAC Mr, GGC GAG ATC M C TTC GCT ACC ATC
ala lys glu leu lys pro leu ala leu lys his lys gly glu ile asn phe dla thr ile
481 / 161 Sll / 171
GAC GCC M G TCC TTC GGC CAG CAC GCT GGC M C CTT M C CTC AAG GTC GGC ACC TGG CCC
asp ala lys ser phe gly gln his ala gly asn leu asn leu lys val gly thr trp pro
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541 / 181 571 t 191
GCT TTC GCT ATC CAG CGC ACC GAG AAG M C GAG AAG TTC CCT ACG M C CAG GAG GCC AAG
ala phe ala ile gln arg thr glu lys asn glu lys phe pro thr asn gln glu ala lys
601 / 201 631 / 211
ATC ACC GAG M G GAG ATT GGC M G TTC GTT GAC GAC TTC CTC GCT GGC M G ATT GAC CCT
ile thr glu lys glu ile qly lys phe val asp asp phe leu ala gly lys ile asp pro
661 /. 221 691 / 231
AGC ATC AAG TCT GAG CCC ATT CCC GAA TCC MT GAC GGT CCC GTA ACT GTC GTC GTT GCC
ser ile lys ser glu pro ile pro glu ser asn asp gly pro val thr val val val ala
721 / 241 751 / 251
CAC M C TAC M G GAT GTC GTC ATT GAC MC GAC M G GAC GTT CTC GTT GAG T~C TAC GCC
his asn tyr lys asp val val ile asp asn asp lys asp val leu val glu phe tyr ala
781 / 261 811 / 271
CCC TGG TGC GGT CAC TGC M G GCT CTT GCT CCC AAG TAC GAG GAG CTC GGC CAG CTC TAC
pro trp cys gly his cys lys ala leu ala pro lys tyr glu glu leu gly gln leu tyr
841 / 281 871 / 291
GCT TCC GAC GAG CTC TCC M G TTG GTG ACC ATT GCC M G GTT GAC GCT ACT CTC M C GAC
ala ser asp glu leu ser lys leu Yal thr ile ala lys Yal asp ala thr leu asn asp
901 / 301 931 / 311
GTT CCC GAC GAG ATC CAA GGT TTC CTA CCA TCA AGC CTC TTC CCG CTG GCA AGA AGG ATG
val pro asp glu ile gln gly phe leu pro ser ser leu phe pro leu ala arg arg met
961 / 321 991 / 331
CCC CAG TCG ACT ACT CTG GTT CCG CAC TGT CGA GGA TCT CGT CCA GTT CAT CGA AGA GAA
pro gln ser thr thr leu val pro his cys arg gly ser arg pro val his arg arg glu
1021 / 341 1051 / 351
CGG CTC ACA CAA GCT AGC GCC AGC GTT GGC G M GCT GTT GAA GAT GCT ACC GAG TCC GCC
arg leu thr gln ala ser ala ser val gly glu ala val glu asp ala thr glu ser ala
1081 / 361 1111 / 371
AAG GCC AGT GCC TCT TCC GCC ACA GAC TCT GCT GCC TCA GCT GTA TCA GAA GGC ACC GAG
lys ala ser ala ser ser ala thr asp ser ala ala ser ala Yal ser glu gly thr glu
1141 / 381 1171 / 391
ACG GTC AAG TCT G~T GCG TCT GTC GCT TCC GAC TCA GCC TCT TCC GCC GCT TCC GAG GCT
thr Yal lys ser gly ala ser val ala ser asp ser ala ser ser ala ala ser glu ala
1201 / 401 1231 / 411
ACC M G TCT GTC M G TCT GCC GCG TCC GAG GTT ACC M C TCT GCC TCG TCG GCT GCG TCA
thr lys ser val lys ser ala ala ser glu val thr asn ser ala ser ser ala ala ser
1261 / 421 1291 / 431
GAG GCT TCA GCT TCG ~CC TCA AGC GTC M G GAC GAG TTG T M
glu ala ser ala ser ala ser ser val lys asp glu leu OCH
CA 02204362 1997-0~-02
Homology searches with Alt a 45 in the SWISSPROT protein
database have revealed that Alt a 45 is a protein disulfide
isomerase (PDI). Multiple alignment with PDI sequences of
several organisms has reflected the high homology of Alt a 45
to PDI sequences. The established consensus shows amino acid
identities straight through all organisms. A central motif of
homology is defined by the sequence "EFYAPWCGHCK." The
function of protein disulfide isomerase (PDI) is support in
the folding process of proteins. The exact mechanism is not
yet known. The active site of PDI is similar to that of
thioredoxin. The high consensus between homologous proteins is
found from bacteria to plants and on up to higher mammals. The
PDI protein is found in the lumen of the endoplasmatic
reticulum.
Sequence 2: Alt a 45: B-cell epitopes
(1) INFORMATION ON SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: Listed individually
(B) TYPE: Protein
(ii) NATURE OF MOLECULE: Peptides
(iii) HYPO~ CAL: No
(v) NATURE OF FRAGMENT: N terminus to C terminus
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Alternaria alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
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Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr Ala Asp Lys (11-25)
Phe Ala Ala Asp Asp Lys Ala Ser Asn Gly Glu Thr Phe Thr ier Val Ala
(32-47)
Ser Pro Ser Thr Thr Ala Arg Thr Ser Ser Pro Arg Pro Ser Het Arg Thr TyrPro Arg Leu Arg Lys Val Ala (79-io3)
Phe Ala Gly Glu Thr Pro Gly Glu Gly Glu Arg Gly Glu Gly Glu Phe Ala LysGly Glu Leu Lys Pro Leu Ala Leu (130-149)
Gln Arg Thr Gly Glu Lys Asn Gly Glu Lys Phe Pro Thr Asn Gln Gly Glu AlaLys Ile Thr Gly Glu Lys Gly Glu Ile Gly Lys Phe Val Asp Asp (185-212)
Asp Pro Ser Ile Lys Ser Gly Glu Pro Ile Pro Gly Glu Ser Asn Asp Gly ProVal Thr (219-236)
Ala Arg Arg Het Pro Gl n 5er Thr Thr Leu Val Pro His Cys (317-330)
Arg Gly Ser Arg Pro Yal His Arg Arg Gly GlU Arg Leu Thr Gln Ala Ser AlaSer Val Gly Gly Glu Ala (331-352)
CA 02204362 1997-0~-02
Sequence 3: Predicted amphipathatic segments = T-cell epitopes
Flags Hidpoints Angles Score
.
K 9:22 85:120 33,4 YSEVTKOTLEEFKT
41:50 85:105 23.4
K P 66:71 125:135 11.9 KAEGVK
P 82:85 115:125 6.3
K P 96:101 115:120 16.0 YPRLRK
P 110:114 80:90 6.8
P 116:120 85:1~5 10.4
K P 141:145 100:125 9.4 AKELK
P 178:182 130:135 8.1
K P 204:216 80:100 31.5 KEIGKFYDDFLAG
K P 257:267 95:110 28.B EFYAP~CGHCK
275:281 110:125 15.2
K 283:293 90:130 25.6 DELSKLVTIAK
P 295:309 85:105 39.9 DATLHDVPDEIQGFL
K 345:361 90:125 45.9 ASASYGEAVEDATESAK
~ 364:3BS B5:135 49.3 ASSATDSAASAYSEGTETYKSG
K 391:413 80:115 73.2 DSASSAASEATKSVKSAASEVTH
(1) INFORM~TION ON SEQ ID NO: 3
(i) SEQUENCE CHARAC~ERISTICS:
(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 alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Val Ser Gly Glu Val Thr Lys Asp Thr Leu Gly Glu Gly Glu Phe Lys Thr
(9-22)
Lys Ala Gly Glu Gly Yal Lys (66-71)
Tyr Pro Arg Leu Arg Lys (96-101)
Ala Lys Gly Glu Leu Lys (141-145)
Lys Gly Glu Ile Gly Lys Phe Val Asp Asp Phe Leu Ala Gly (204-216)
Gly Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys (257 267)
Asp Gly Glu Leu Ser Lys Leu Val Thr Ile Ala Lys (283-293)
Asp Ala Thr Leu Asn Asp Val Pro Asp Gly Glu lle Gln Gly Phe Leu
(2g5-309)
Ala Ser Ala Ser Val Gly Gly Glu Ala Val Gly Glu Asp Ala Thr Gly Glu Ser
Ala Lys (345-361)
Ala Ser Ser Ala Thr Asp Ser Ala Ala Ser Ala Val Ser Gly Glu Gly Thr Gly
Glu Thr Val Lys Ser Gly (364-385)
Asp Ser Ala Ser Ser Ala Ala Ser Gly Glu Ala Thr Lys Ser Val Lys Ser Ala
Ala Ser Gly Glu Yal Thr Asn (391-413)
CA 02204362 1997-0~-02
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 proline (P) (= flags).
Potential T-cell epitopes are present only when the score
index is greater than 10.
B: Alt a 12
The following sequence 4 shows the complete cDNA sequence
of Alt a 12 and of the amino acid sequence derived therefrom.
The open reading frame comprises 333 bp or 111 amino acids.
The calculated molecular weight is 11728 daltons and thus
correlates with the antigenic protein observed at 45 kD, which
is recognized in the Western blot of 8% of the patients.
Sequence 4: Alt a 12 11728 daltons
(1) INFORMATION ON SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs/111 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 alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
DNA sequence 333 b.p. ATGTCTACCTCC ... CTCTTCGACTAA linear
1 / 1 31 / 11
ATG TCT ACC TCC GAG CTC GCC ACC TCT TAC GCC GCT CTC ATC CTC GCT GAT GAC GGT GTC
~et ser thr ser glu leu ala thr ser tyr ala ala leu ile leu ala asp asp gly val
CA 02204362 1997-0~-02
\
,
61 ~ 21 91 / 31
~C ATC AC~ GCC GAC MG C~ CM TCC CTC ATC MG GCC GCA AAG ATC. G~ G GrC G~G
asp ile thr ala asp lys leu gln ser leu ile lys ala ala lys ile ~lu glu val glu
121 ~ 41 151 / 51
CCC ATC TGG ACG ACC CTG rrc GCC MG GCT CrT GAG GGC MG G~T GrC MG G~C ~G ~A
pro ile trp thr thr leu phe ala lys ala leu glu gly lys asp val lys asp leu leu
181 / 61 211 / 71
CTG MC GTC GGC TCA GGC GGC GGT GCr GCC CCG CTG CCG G~G gCg cTG crc CTG CGC TG~
leu asn val gly ser gly gly gly ala ala pro leu pro glu ala leu leu leu arg trp
241 / 81 271 / 91
CGT GCI GCT G~T GCC GCA CCA GCT GCr G4G G4G MG MG G~ G MG GAG GAG TCG
arg ala ala asp ala ala pro ala ala glu glu lys lys glu glu glu lys glu glu ser
301 / 101 331 / 111
64C G~G G~C ATG GGC TTC GGT crc rrc G~C TM
asp glu asp net gly phe gly leu phe asp OCH
Here homology searches in the SWISSPROT protein database
revealed homologies to ribosomal proteins. The allergenic
protein Alt a 12 is of interest not only because of its
property as an allergen of Alternaria alternata. Ribosomal
proteins, here especially the human ribosomal proteins Pl and
P2, have been described in the literature as autoantigens
(Francoeur et al., 1985, Rich et al., 1987, Hines et al.,
1991). 20% of patients with lupus erythematosus have
autoantibodies (anti-rRNP) to components of ribosomes, in
particular autoantibodies to the ribosomal proteins PO (38
kD), Pl (16 kD) and P2 (15 kD). Human autoantibodies cross-
react with similar proteins, which means that epitopes that
have been strongly preserved in evolution are recognized. The
basis of immunologic cross-reactivity is provided by the C-
terminal 17-amino-acid group region KEESEESD(D/E)DMGFGLFD.
Whether sensitization by Alt a 12 which has taken place in
childhood and youth correlates with an autoimmune disease
appearing in adulthood needs precise PX~;n~tion. However,
administration of ribosomal proteins failed to produce any
CA 02204362 1997-0~-02
,
16
autoimmune disease in mice (Hines et al., 1991). Other
ribosomal proteins (Cla h 11 and Alt a 11) likewise have
already been identified as allergens (Achatz et al., 1994).
The B-cell epitopes shown in the next sequence 16 were
calculated taking secondary structure, surface position,
hydrophilicity, flexibility, etc. into consideration.
Sequence 5: Alt a 12: B-cell epitopes
(1) INFORMATION ON SEQ ID NO: 5
(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 alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Ala Asp Asp Gly Val Asp (16-21)
Thr Ala Asp Lys Leu Gln Ser Leu (23-30)
Ala Lys Ile Gly Glu Gly Glu Val Gly GlU Pro Ile Trp Thr (34-44)
Ala Leu Gly GlU Gly Lys Asp Val Lys Asp (50-58)
Val Gly Ser Gly Gly Gly Ala Ala (63-70)
Trp Arg Ala Ala Asp Ala (80-85)
Ala Pro Ala Ala Gly Glu Gly Glu Lys Lys Gly GlU Gly GlU Gly GlU Lys Gly
Glu Gly Glu Ser Asp Gly Glu Asp Het Gly (105)
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17
Sequence 6: Predicted A rhir~thatic segments = T-cell epitopes
Fl~g~ ~idpo~nt~ Anqle~ score
_ _ _ _ _ _ _ _ _ _ _ _ _
K P 26:37 90:135 27.6 KLaSLIKAAKIE
K 39:49 80:115 22.0 YEPII~TTLFAK
K 52:55 80:135 6.4
(1) INFORMATION ON SEQ ID NO: 6
(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 alternata
(C) DEVELOPMENTAL STAGE: Spores and vegetative hyphae
Lys Leu Gln Ser Leu lle Lys Ala .Ila Lys lle Gly Glu (26-37)
Yal Gly Glu Pro lle Trp Thr Thr ,eu Phe Ala Lys (39-49.)
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 proline (P). Potential T-
cell epitopes are present only when the score index is greater
than 10.
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18
6 . Ref erences
Achatz. G., Oberkofler, H., Lechenauer, E., Simon, B., Unger, A., Kandler, D.,
Ebner, C., Prillinger, H., Kraft, D., Breit~nbach~ M. (1994).
Molecular cloning of major and minor allcrgens of Alternaria alternata and
Cladosporium herbarum.
Mol. Immunol. in press.
Birkner, T., Rumpold, H., Jarolim, E. Ebner, H., Breitenbach, M., Skarvil, F.,
Scheiner, O., Kraft, D. (19g0).
Evaluation of immunotherapy-induces changes in specific IgE, IgG and IgG subclasses
in birch pollen allergic patients by means of immunoblotting. Correlation with clinical
response.
Allergy 45, 418.
Bousquet, J., Becker, W.M., He~aoudi, A. (1991).
Differences in clinical and immunologic reactivity of patients allergic to grass pollens
and to multiple-pollen spe~ies. II. Efficacy of a double blind, placebo-controlled,
specific immunotherapy with standardized extracts.
J. Allergy Clin. Immunol. 88, 43.
Francoeur, A.M., Pe~bles, C.L., He~krn~n, K.J., Lee, J.C.. Tan, E.M. (1985).
Identification of ribosomal protein autoantigens.
J. Immunol. 135,1767.
Hines, J.J., Wei~sbach, H., Brot, N., Elkon, K. (1991).
Anti-P ~lto~ntibody production requires Pl/P2 as immunogens but is not driYen byexogenous self-antigen in mrl mice.
J. Immunol.. 146, 3386
Margalit, H., Spogue, J.L., Cornette, J.L., Cease, K.B., Delisi, C., Berzofsky, J.A.
(1987).
Prediction of immunodominant Helper T cell antigenic sites from the primary
sequence.
J. Immunol. 138, 2213.
Rammensee, H.G.. Falk, K., Rotzschke. 0. (1993).
CA 02204362 1997-05-02
19
MHC molecules as peptide receptors.
Current Opinion in Immunol. 5. 35.
Rich, B.E., Steitz, J.A. (19~7).
Human acidic ribosomal phosphoproteins P0, Pl and P2: analysis of cDNA clones, in
vitro synthesis and asscmbly.
Mol. Cell. Biol. 7, 4065.
Rothbard, J.B., Gef~er, M.L. (1991).
Interactions betwcen immunogcnic peptides and MHC proteins.
Ann. Rev. Immunol. 9, 527.
Sanger, F., Niclclen, S., Coulson. A.R. (1977).
DNA sequencinE with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA 74, 5463-5468
