U.S. patent application number 12/381719 was filed with the patent office on 2009-11-26 for recombinant soluble fc receptors.
Invention is credited to Robert Huber, Uwe Jacob, Peter Sondermann.
Application Number | 20090292113 12/381719 |
Document ID | / |
Family ID | 8233085 |
Filed Date | 2009-11-26 |
United States Patent
Application |
20090292113 |
Kind Code |
A1 |
Sondermann; Peter ; et
al. |
November 26, 2009 |
Recombinant soluble Fc receptors
Abstract
Recombinant soluble Fc receptors according to the present
invention are characterized by the absence of transmembrane
domains, signal peptides and glycosylation. Such Fc receptors can
easily be obtained by expressing respective nucleic acids in
prokaryotic host cells and renaturation of the obtained inclusion
bodies, which procedure leads to a very homogenous and pure
product. The products can be used for diagnostic as well as
pharmaceutical applications and also for the generation of crystal
structure data. Such crystal structure data can be used for the
modelling of artificial molecules. A further embodiment comprises
coupling the Fc receptors according to the invention to solid
materials like chromatography materials that can be used to
separate and/or enrich antibodies.
Inventors: |
Sondermann; Peter;
(Krailling, DE) ; Huber; Robert; (Germering,
DE) ; Jacob; Uwe; (Munchen, DE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
8233085 |
Appl. No.: |
12/381719 |
Filed: |
March 16, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11327695 |
Jan 6, 2006 |
7504482 |
|
|
12381719 |
|
|
|
|
09856933 |
Feb 27, 2002 |
7074896 |
|
|
PCT/EP99/09440 |
Dec 3, 1999 |
|
|
|
11327695 |
|
|
|
|
Current U.S.
Class: |
530/391.7 ;
530/350; 703/11 |
Current CPC
Class: |
A61P 19/02 20180101;
A61K 38/00 20130101; C07K 14/70535 20130101; C07K 2299/00 20130101;
A61P 31/18 20180101; A61P 29/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
530/391.7 ;
530/350; 703/11 |
International
Class: |
C07K 19/00 20060101
C07K019/00; C07K 14/00 20060101 C07K014/00; G06G 7/48 20060101
G06G007/48 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 1998 |
EP |
98 122 969.3 |
Claims
1-40. (canceled)
41-66. (canceled)
67. A purified, crystalline, recombinantly produced, soluble
Fc.gamma. or Fc.epsilon. receptor molecule, which lacks any of (i)
a transmembrane domain, (ii) signal peptide, and (ii)
glycosylation.
68. The purified, crystalline recombinantly produced soluble
Fc.gamma. or Fc.epsilon. receptor molecule of claim 67, which is a
human receptor.
69. The purified, crystalline, recombinantly produced soluble
Fc.gamma. or Fc.epsilon. receptor molecule of claim 67, comprising
an amino acid sequence set forth in SEQ. ID NO. 3 or SEQ ID NO:
4.
70. A purified complex of the purified, crystalline recombinantly
produced soluble Fc.gamma. or Fc.epsilon. receptor of claim 1, and
immunoglobulin.
71. The purified, crystalline, recombinantly produced soluble
Fc.gamma. or Fc.epsilon. receptor molecule of claim 67, produced by
expression of said molecule in a prokaryotic cell.
72. A method of identifying an Fc.gamma. or Fc.epsilon. receptor
inhibitor of claim 67, comprising inputting, crystal structure data
from said receptor into a computer modeling program to identify an
inhibitor which is complementary to said receptor.
73. A method for identifying or preparing an Fc.gamma. or
Fc.epsilon. receptor molecule of claim 67, wherein said inhibitor
is complementary to said molecule, comprising inputting crystalline
data from said molecule into a computer aided modeling program to
identify or to prepare said inhibitor.
74. A method for identifying or preparing an inhibitor of the
molecule of claim 67, comprising: (i) obtaining three dimensional
structural data for said molecule, and (ii) selecting or designing
one of: (a) an inhibitor of said molecule which is complementary to
an Fc.gamma. or Fc.epsilon. receptor binding site of an
immunoglobulin; (b) an inhibitor which is complementary to an
immunoglobulin binding site of an Fc.gamma. or Fc.epsilon.
receptor, and (c) an antagonist or competitive Fc.gamma. or
Fc.epsilon. receptor.
75. A method for modulating interaction of an Fc receptor and an
immunoglobulin molecule, comprising contacting a mixture of said Fc
receptor and said immunoglobulin molecule with an inhibitor
identified by the method of claim 73 or 74, under conditions
favoring modulating interaction.
76. The method of claim 75, wherein said inhibitor partially or
completely inhibits binding and said immunoglobulin.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 11/327,695 filed Jan. 6, 2006, which is a
divisional application of U.S. application Ser. No. 09/856,933
filed Feb. 27, 2002, now U.S. Pat. No. 7,074,896, which is a .sctn.
371 of PCT/EP99/09440, filed Dec. 3, 1999, incorporated herewith by
reference in its entirety.
[0002] The present invention relates to recombinant soluble Fc
receptors (FcR), recombinant nucleic acids coding for such Fc
receptors, host cells containing corresponding nucleic acids as
well as a process for the determination of the amount of antibodies
of a certain type contained in the blood, plasma or serum of a
patient, a process for the determination of the immune status of
patients with chronic diseases of the immune system and a process
for the screening of substances in view of their ability to act as
inhibitors of the recognition and binding of antibodies to the
respective cellular receptors. Further, the present invention is
concerned with pharmaceutical compositions containing the
recombinant soluble FcRs, crystalline preparations of FcRs and
FcR/Ig-complexes and especially of the use of such crystalline
preparation for the generation of crystal structure data of Fc
receptors as well as FcR inhibitors and pharmaceutical compositions
containing such FcR inhibitors.
[0003] A still further subject of the present invention is a
recombinant Fc receptor coupled to a solid phase, e.g. a
chromatography carrier material. The use of such chromatography
material, which is another subject of the present invention, lies
in the absorption of immunoglobulins from a body fluid of patients
or from culture supernatants of immunoglobulin producing cells.
[0004] Fc receptors (FcRs) play a key role in defending the human
organism against infections. After pathogens have gained access to
the blood circulation they are opsonized by immunoglobulins (Igs).
The resulting immunocomplexes bind due to their multivalency with
high avidity to FcR bearing cells leading to clustering of the
FcRs, which triggers several effector functions (Metzger, H.,
1992A). These include, depending on the expressed FcR type and
associated proteins, endocytosis with subsequent neutralization of
the pathogens and antigen presentation, antibody-dependent cellular
cytotoxity (ADCC), secretion of mediators or the regulation of
antibody production (Fridman et al, 1992; van de Winkel and Capel,
1993).
[0005] Specific FcRs exist for all Ig classes, the ones for IgG
being the most abundant with the widest diversity. Together with
the high affinity receptor for IgE (Fc.epsilon.Rla), Fc.gamma.RI
(CD64), Fc.epsilon.RII (CD32) and Fc.epsilon.RIIIa (CD16) occur as
type I transmembrane proteins or in soluble forms (sFcRs) but also
a glyeosylphosphatidylinositol anchored form of the Fc.epsilon.RIII
(Fc.epsilon.RIIIb) exists. Furthermore, Fc.epsilon.Rs occur in
various isoforms (Fc.epsilon.RIa, b1, b2, c; Fc.epsilon.RIIa 1-2,
b1-3, c) and alleles (Fc.epsilon.Rlla1-HR, -LR; Fc.epsilon.RIIIb-NA
1, -NA2) (van de Winkel and Capel, 1993). In contrast to the
overall homologous extracellular parts, the membrane spanning and
the cytoplasmic domains differ. They may be deleted entirely or be
of a size of 8 kDa. They may contain either a 26 amino acid
immunoreceptor tyrosine-based activation motif (ITAM) as in
Fc.gamma.RIIa or a respective 13 amino acid inhibitory motif (ITIM)
in Fc.gamma.RIIb involved in signal transduction (Amigorena et al,
1992).
[0006] Judged by the conserved spacing of cysteins, the
extracellular part of the FcRs consists of three (Fc.gamma.RI,
CD64) or two (Fc.epsilon.RI, Fc.gamma.RII, CD32 and Fc.gamma.RIII,
CD16) ig-like domains (10 kDa/domain) and therefore belongs to the
immunoglobulin super family. These highly glycosylated receptors
are homologues, and the overall identity in amino acid sequence
among the Fc.gamma.Rs and Fc.epsilon.RIa exceeds 50% in their
extracellular regions. Nevertheless, the affinity of FcRs to their
ligands varies widely. The higher affinity of
.apprxeq.10.sup.8M.sup.-1 of the Fc.gamma.RI to Fc-fragment is
assigned to its third domain, while the other Fc.gamma.Rs with two
domains have an affinity to IgG varying between 10.sup.5 and
10.sup.7M.sup.-1. The affinity of the two domain Fc.epsilon.Rla to
IgE exceeds these 30 values by far with a constant of
10.sup.10M.sup.-1 (Metzger, H., 1992B). In contrast to the
mentioned FcRs the low affinity receptor for IgE Fce-RII represents
a type transmembrane protein and shows a lower homology.
[0007] Fc.gamma. Rs are expressed in a defined pattern on all
immunological active cells. Fc.gamma.RI is constitutively expressed
on monocytes and macrophages and can be induced on neutrophils and
eosinophils. The physiological role of Fc.gamma.RI is still unknown
as the expression on monocytes is not vital (Ceuppens et al.,
1988). The GPI anchored form of Fc.gamma.RIII (Fc.gamma.RIIIb) is
exclusively expressed on granulocytes. Due to its missing
cytoplasmic part, the signal transduction into the cell occurs
solely via other transmembrane proteins like complement receptor
type 3 (CR3) that can at least associate with Fc.gamma.RIIIb (Zhou
et al, 1993; Poo et al, 1995). Fc.gamma.RIIIa is mainly expressed
on monocytes and macrophages but only in conjunction with
associated proteins (e.g. .alpha.- or .gamma.-chains). Fc.gamma.RII
is the receptor with the widest distribution on immunocompetent
cells and is mainly involved in the endocytosis of
immunocomplexes.
[0008] Fc.gamma.RIIa and Fc.gamma.RIIb differ in their
extracellular region by only 7% of the amino acid residues.
Nevertheless, both forms can be distinguished by their binding
characteristics to human and mouse IgG subclasses (van de Winkel
and Capel, 1993) and their differing affinity to human IgGs
(Sondermann et al., 1998A). The situation is rendered even more
complicated by the high responder/low responder (HR/LR)
polymorphism of Fc.gamma.RIIa named after the ability of T cells
from some individuals to respond to murine IgG1-induced mitogenesis
(Tax et al, 1983). Later, it was found that the two exchanges in
the amino acid sequence between the LR and the HR form modify the
ability to bind human IgG2, which leads to the suggestion that at
least one of them is involved in IgG binding (Hogarth et al,
1992).
[0009] In contrast to the beneficial role FcRs play in the healthy
individual, they also transmit the stimulation of the immune system
in allergies (Fc.epsilon.RIa) or autoimmune diseases. Moreover,
some viruses employ Fc.gamma.Rs to get-access to cells like HIV
(Homsy et al, 1989) and Dengue. (Littaua et al, 1990) or slow down
the immune response by blocking Fc.gamma.Rs as in the case of Ebola
(Yang et al, 1998) and Measles (Ravanel et al, 1997).
[0010] Hence, the object underlying the present invention was to
provide receptors which are easy to produce and can advantageously
be used for medical or diagnostic applications. Moreover, it was an
object of the invention to provide soluble receptors exhibiting a
binding specificity and activity which is analogous to that of the
receptors occurring naturally in the human body and which,
additionally, make it possible to produce crystals suitable for a
structure determination.
[0011] This object is accomplished by recombinant soluble Fc
receptors which consist only of the extracellular portion of the
receptor and are not glycosylated. The receptors according to the
present invention are therefore characterized by the absence of
transmembrane domains, signal peptides and glycosylation.
[0012] Particularly preferred for the present invention are
Fc.gamma. or Fc.epsilon. receptors. This is because IgG and IgE
molecules are characteristic for a multiplicity of diseases and
conditions, so that their determination and possible ways of
influencing them are of great interest. FIGS. 11 and 12 show an
alignment of amino acid sequences of the extracellular parts of
some Fc.gamma.Rs and Fc.epsilon.RI. The FcRs according to the
invention include all these sequences or parts thereof that still
retain binding capacity to antibodies and/or proper
crystallization.
[0013] In a particularly preferred embodiment of the invention the
recombinant soluble FcR is a Fc.gamma.RIIb receptor. Further, it is
particularly preferred that the receptor be of human origin. In a
particularly preferred embodiment, it contains an amino acid
sequence as shown in one of SEQ ID NO: 1 to SEQ ID NO:6.
[0014] According to the present invention, the preparation of the
soluble Fc receptors preferably takes place in prokaryotic cells.
After such expression, insoluble inclusion bodies containing the
recombinant protein form in prokaryotic cells, thus facilitating
purification by separation of the inclusion bodies from other cell
components before renaturation of the proteins contained therein
takes place. The renaturation of the FcRs according to the present
invention which are contained in the inclusion bodies can
principally take place according to known methods. The advantage of
the preparation in prokaryotic cells, the production of inclusion
bodies and the thus obtained recombinant soluble Fc receptors make
it possible to obtain a very pure and, in particular, also very
homogeneous FcR preparation. Also because of the absence of
glycosylation the obtained product is of great homogeneity.
[0015] Soluble Fc receptors hitherto produced by recombinant means
particularly exhibited the disadvantage that a much more elaborate
purification was required, since they were expressed in eukaryotic
cells and, due to the glycosylation which is not always uniform in
eukaryotic cells, these products were also less homogeneous.
[0016] The recombinant soluble Fc receptors according to the
present invention even make it possible to produce crystals
suitable for use in X-ray analysis, as shall be explained later on
in the description of further embodiments of the invention. The
FcRs of the present invention moreover exhibit practically the same
activity and specificity as the receptors naturally occurring in
vivo.
[0017] A further subject matter of the present invention is a
recombinant nucleic acid having a sequence coding for a recombinant
soluble Fc receptor according to the present invention.
[0018] The nucleic acid according to the present invention may
contain only the coding sequences or, additionally, vector
sequences and/or, in particular, expression control sequences
operatively linked to the sequence encoding the recombinant FcR,
like promoters, operators and the like.
[0019] In a particularly preferred embodiment the nucleic acid of
the present invention contains a sequence as shown in one of SEQ ID
NO:7 to SEQ ID NO: 12. For a comparison, SEQ ID NO: 13 and SEQ ID
NO: 14 show the respective wild type sequences coding for
Fc.gamma.RIIb and Fc.epsilon.RIa. SEQ ID NOs: 15-18 show the wild
type sequences for Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIII and
Fc.epsilon.RII.
[0020] If the nucleic acid of the present invention contains vector
sequences, then these are preferably sequences of one or several
prokaryotic expression vectors, preferably of pET vectors. Any
other known functions or components of expression vectors may also
be contained in the recombinant nucleic acid according to the
present invention if desired. These may, for instance, be
resistance genes allowing for an effective selection of transformed
host cells.
[0021] A still further subject matter of the present invention is a
host cell containing a recombinant nucleic acid according to the
present invention. As repeatedly mentioned above, the host cell
preferably is a prokaryotic host cell, particularly an E. coli
cell.
[0022] The recombinant soluble Fc receptors according to the
present invention can be used for a multitude of examinations or
applications because they specifically react with antibodies. In
vivo, the soluble Fc receptors are powerful immunoregulators which,
if present in elevated levels, result in a remarkable suppression
of the immune system which leads to many partly known and partly
not yet understood effects. Based on these effects, several
applications of the Fc receptors according to the present invention
are further subject matters of the present invention.
[0023] One such subject is a process for the determination of the
amount of antibodies of a certain type in the blood or serum of a
patient, which is characterized by the use of a recombinant soluble
FcR according to the invention in an immunoassay, and the
determination of the presence of FcR-antibody complexes. Such assay
allows to screen for the presence of a certain kind of antibody and
allows also for the determination of the amount of antibodies
present in the blood, plasma or serum of a patient.
[0024] Any type of immunoassay is principally suitable for the use
according to the present invention, as long as the presence of
FcR-antibody complexes can thereby be detected. Both ELISA
(enzyme-linked immunosorbent immunoassay), particularly sandwich
assays, and RIA (radio-immunoassay) are suitable, but also
competitive testing methods. In a preferred embodiment of the
invention where the presence and/or the amount of IgE antibodies is
to be examined, an Fc.epsilon.R is used as recombinant soluble
receptor according to the present invention. In particular, this
method is suited and advantageous for determining a predisposition
or manifestation of an allergy.
[0025] Moreover, a method is preferred in which the presence of
soluble FcRs is to be determined and, if required, quantified. For
such determination preferably a competitive immunoassay method is
used, wherein as competition reagent a recombinant soluble receptor
according to the invention is used, most preferably a recombinant
Fc.gamma.R. By means of this test among others the immune status of
patients with chronic diseases of the immune system can be
determined in a competitive immunoassay. Chronic diseases in the
sense of these processes are for instance AIDS, SLE (systemic lupus
erythematosus), MM (multiple myeloma) or rheumatoid arthritis, or
in the case of Fc.epsilon.RII in .beta.-CLL (Gordon et al., 1987),
hyper IgE syndrome (Sarfati et al., 1988) or HCL (Small et al.,
1990).
[0026] A further advantageous use of the recombinant receptor
according to the present invention lies in the screening of
substances in view of their ability to act as inhibitors of the
recognition and binding of antibodies to the respective cellular
receptors.
[0027] By means of modern screening techniques such as HTPS (high
throughput screening) in combination with multi-well microtiter
plates and automatic pipetting apparatuses it is nowadays possible
to simultaneously test a multitude of substances for specific
properties. As the FcRs according to the present invention can be
easily produced at low cost, they can also be used in such series
tests by which substances having an inhibiting effect can easily be
identified.
[0028] Particularly preferred is such use according to which Fc
receptors according to the present invention are used to find or
screen inhibitors capable of inhibiting the recognition and binding
of the respective antibodies to the particular receptor of
interest.
[0029] A further area of application of the substances according to
the invention lies in the pharmaceutical field. Hence, a further
subject matter of the invention is a pharmaceutical composition
comprising as active agent a recombinant soluble FcR according to
the invention. According to the present invention, this
pharmaceutical composition may of course comprise conventional
useful carrier and auxiliary substances. Such substances are known
to the person of skill in the art, the mode of administration also
having to be taken into account. The pharmaceutical composition of
the present invention can be advantageously used for the treatment
or prevention of autoimmune diseases, allergies or tumor
diseases.
[0030] Soluble forms of Fc receptors such as Fc.gamma.RIII mediate
isotype-specific regulation of B cell growth and immunoglobulin
production. In a murine model of myeloma, sFcR suppresses growth
and immunoglobulin production of tumor cells (Muller et al, 1985;
Roman et al, 1988; Teillaud et al, 1990). Furthermore, sFcR binds
to surface IgG on cultures of human IgG-secreting myeloma cells and
effects suppression of tumor cell growth and IgG secretion.
Prolonged exposure of these cells to sFcR results in tumor cell
cytolysis (Hoover et al, 1995).
[0031] Also, overreactions of the immune system in allergic
reactions or due to massive antigen load might be reduced by, for
example, intravenous application of soluble FcR (Lerino et al,
1993).
[0032] Therefore, a preferred pharmaceutical composition according
to the invention for use in the treatment of AIDS, rheumatoid
arthritis or multiple myeloma contains a recombinant soluble
Fc.gamma. receptor and, preferably, a receptor having the amino
acid sequence as shown in SEQ ID NO: 1-4.
[0033] It was also of great interest to obtain crystal structure
data of Fc receptors and/or Fc receptor/Ig complexes. On the one
hand, these are a key to the understanding of molecular mechanisms
in immunocomplex recognition. On the other hand, these structural
data can be used to find out common features in the structures of
different Fc receptors and use the knowledge of the structures to
generate inhibitors or identify and produce new artificial antibody
receptors.
[0034] It was also of great interest to obtain information on the
concrete binding sites of immunoglobulins to their respective
receptors in naturally occurring three-dimensional molecules.
Therefrom even more precise findings on the interactions between
antibody and receptor can be obtained and also on how these
interactions can be modulated. In this connection modulation means
either an enhancement of the interaction or a reduction leading to
an inhibition by e.g. covering the binding sites on one or more
parts of the complex.
[0035] To obtain such crystal structure data and conformation
information, a crystalline preparation of the recombinant soluble
Fc receptor according to the invention is used. The recombinant
soluble FcRs according to the invention surprisingly can be
obtained pure enough to produce crystals that give reliable X-ray
structure determination data. Such crystallization was not possible
with the hitherto produced receptor molecules, mostly due to their
lack of homogeneity.
[0036] Therefore, another embodiment of the present invention
concerns a crystalline preparation of an Fc receptor according to
the invention. Yet another embodiment of the present invention is a
crystalline preparation of a complex of soluble Fc receptor
according to the invention together with the related immunoglobulin
Fc part. Particularly preferred embodiments are shown in the
examples as well as the relevant crystal structure data. Via
crystal structure analysis of the crystalline preparations the
exact amino acids of the Fc receptor/Ig complexes could be detected
which mediate the coupling. These amino acids are in shown FIGS. 6a
and 6b and the type of binding between the individual amino acids
of both molecules in the complex is also indicated. A further
embodiment of the present invention is therefore the use of a
crystalline preparation of a recombinant soluble Fc receptor for
the generation of crystal structure data of Fc receptors. From this
crystal structure data information about the three-dimensional
structure and the active sites for the binding of antibodies can be
obtained. Especially preferably is the use of a crystalline
preparation of a complex of recombinant soluble Fc receptor
according to the invention and the corresponding immunoglobulin
molecule for the generation of crystal structure data for the
complexes. These data allow to determine the actual interactions
that are formed between the two molecules and allow for the first
time to obtain exact information about the interaction of the
molecules thereby conferring knowledge about possible sites for
inhibition or enhancement of the binding. On the basis of the
information obtained from the crystal structure data the findings
necessary for effecting modulation of the interaction between Fc
receptor and immunoglobulin can be obtained. This modulation can be
range from enhancement to complete inhibition to an inhibition of
the binding.
[0037] The stated applications are merely preferred embodiments of
the use of the crystal structure data. Many other applications seem
possible, too.
[0038] Suitably, the structural data for the generation and/or
identification of inhibitors or new receptors, respectively, are
used in a computer-aided modelling program.
[0039] Particularly preferred for the present invention are the
structures of FcRs or FcR:Fc-fragment complexes as exemplified in
figures and examples. Such structures can be used to design
inhibitors, antagonists and artificial receptor molecules.
[0040] Computer programs suitable for computer-aided drug design
and screening are known to the person skilled in the art and
generally available. They provide the possibility to examine
umpteen compositions on the computer in view of their ability to
bind to certain molecule when the corresponding structure dates are
entered in the computer. With the help of this possibility a great
number of known chemical compositions can be examined regarding
their inhibiting or antagonistic effect. The person skilled in the
art merely requires the crystal structure dates provided by the
present invention and a commercially available screening program
(Program Flexx: From the GMD-German National Research Center for
Information Technology, Schloss Birlinghoven, D-53754 Sankt
Augustin, Germany). A preferred embodiment of the present invention
therefore is the use of the crystal structure data obtained for the
recombinant soluble Fc receptor according to the invention and for
the complexes of recombinant soluble Fc receptor according to the
invention and corresponding immunoglobulin in a computer aided
modelling program for the identification and production of Fc
receptor inhibitors.
[0041] Likewise, a further embodiment of the present invention is
the use of the crystal structure data obtained for the receptors
according to the invention and the receptor/immunoglobulin
complexes, respectively for the identification and preparation of
new Fc receptors which can be used, e.g. as antagonists and
competitors. The crystal structure data and the data on the amino
acids involved in the binding to Fc receptors obtained therefrom
can serve for example to generate mutated immunoglobulins which can
also be used as inhibitors. It is imaginable that mutated or
chemically modified inhibitors undergo tight binding and thus
effect a blocking of receptors. On the other hand, the data
obtained for the binding sites of immunoglobulins can also be used
for the identification and/or preparation of inhibitors for
immunoglobulin molecules. Since the present invention teaches the
binding sites to the receptor, it is easy to effect a blocking of
the binding sites with the help of relatively simple molecules.
Therefore, a further subject matter of the present invention is the
use of the crystal structure data obtained for the FcR/Ig complexes
for the identification and/or preparation of immunoglobulin
inhibitors.
[0042] Accordingly, still further subject matter of the present
invention are FcR inhibitors which have a three-dimensional
structure which is complementary to the recombinant soluble FcR
according to the invention and inhibit the binding of antibodies to
FcRs.
[0043] Another further subject of the present invention are
immunoglobulin inhibitors which have a three-dimensional structure
which is complementary to the immunoglobulin binding site for
recombinant soluble Fc receptors according to the invention and
inhibit the binding of immunoglobulins to Fc receptors.
[0044] The term "complementary" is to be understood within the
framework of the invention in such a way that the inhibitor
molecules must be substances which are able to cover at least so
many binding sites on the immunoglobulin or on the Fc receptor that
the binding between Fc receptor and immunoglobulin is at least
decisively weakened. Covering can take place both by binding to the
amino acids mediating the complex formation of either component but
also in such a way that at least complex formation is no longer
possible, be it by sterically inhibition or by binding to adjacent
amino acids, however, covering the amino acid involved in the
complex binding between Fc receptor and immunoglobulin.
[0045] In connection with the present invention it was possible for
the first time to determine the exact binding sites and the amino
acids involved in the binding of the antibody and antibody receptor
molecules. One is now able to design specifically binding molecules
and to screen candidate compositions on the computer. This enables
the selection of such compositions from a variety of possibly
candidate compositions which can effect a sufficient inhibition of
complex formation between Fc receptor and immunoglobulin.
[0046] What is important for the inhibitors of the invention is
that, owing to their structure and specificity, they are capable of
binding to the FcRs or immunoglobulins and thus prevent the normal
binding between FcRs and the constant parts of antibodies.
[0047] Preferably, such FcR or IgG inhibitors are small organic
molecules which can easily be administered orally. They are an
interesting alternative to cortisone in the treatment of autoimmune
diseases and host/graft rejections. Such a molecule would also
suppress reinfection rates with certain viruses, e.g. Dengue virus
where the antibody coated virus is Fc.gamma.RIIb dependent
internalized (Littaua et al, 1990), HIV where on CD4. positive T
cells an antibody enhancement of HIV infection is mediated by
Fc.gamma.RIII (Homsy et al, 1989), or Ebola where the virus
secreted glycoprotein inhibits early neutrophil activation by
blocking sFc.gamma.RIII which affects the host response to
infection (Yang et al, 1998).
[0048] The development of inhibitors also leads to substances that
interfere with the recognition of IgE by their receptors. From the
modelled structure of Fc.epsilon.RI, peptides have already been
developed which inhibit mast cell degranulation in vitro. With the
now available knowledge of the structures of the homologue
receptors and the receptor-antibody complex in atomic detail, a new
possibility for a rational drug design is opened.
[0049] The Fc-receptor bind between the two CH2-domains of the
Fc-fragment in the so-called lower hinge region (FIG. 8). The
binding region of the Fc-receptor is described in Example 1 (The
contact interface to IgG). The residues promoting the interaction
between FcR and immunoglobulin are shown in FIGS. 7, 10a and 10b.
Thereby three interaction regions become evident (FIG. 5).
1st Region: FcR (Residues 85 to 87 and Residue 110)-Ig (Chain A
Residues 326-328)
[0050] Proline 328 of the Ig is clamped by the residues Trp 87 and
110 in a sandwich like manner. These residues are conserved among
the IgG and IgE receptors as well as in the IgG and IgE. An
inhibitor binding to this prominent region would strongly interfere
with binding. This region is additionally attractive for inhibitor
design because the exposed hydrophobic surface region comprising
the residues Trp 87, Ile 85, Gly 86 of the receptors could be
employed to obtain additional binding energy. The functional groups
of Thr 113 and Glu 18 and Lys 19 side chains in the vicinity may
contribute especially to specific inhibitor binding.
2nd Region: FcR (Residues 126-132 and Residues 155-158)-Ig (Chain A
and Chain B Residues 234-239)
[0051] The amino terminal residues 234-239 of both Ig chains are
recognised differently by the FcR, thereby breaking the 2-fold
symmetry of the Fc fragment.
[0052] This residues of Fc-fragment chain A are in contact with
residues Val 155-Lys 158 of the receptor and the same residues from
Fc-fragment chain B with receptor residues Gly 126-His 132. This
region shows the most differences in the sequence alignment of the
receptors as well as the immunoglobulins and should therefore be
involved in specificity generation. This deep cleft between the
Fc-fragment chains is well suited for inhibitor design and would be
the site of choice for the development of inhibitors when issues of
specificity are concerned.
3rd Region: FcR (Residues 117, 126 and 129-132)-Ig (Chain B
Residues 264-265 and Residues 296-297)
[0053] This binding region is characterised by a clustering of
amino acid residues carrying functional groups in their side
chains, that might be employed in various ways for inhibitor design
on the receptor and the Ig side of the contact.
[0054] Molecules that interact with one or more of the above
described regions, and are designed or screened explicitly for
exploiting the knowledge of binding sites are considered as
inhibitors according to the invention.
[0055] Further subject matters of the present invention are
pharmaceutical compositions containing as active agent an FcR
inhibitor or an immunoglobulin inhibitor as mentioned above. Such
pharmaceutical compositions may, for example, be used in the
treatment or prevention of diseases which are due to overreactions
or faulty reactions of the immune system, preferably the treatment
or prevention of allergies, autoimmune diseases or anaphylactic
shock.
[0056] A further subject of the present invention is the sFcR
according to the invention, bound to a solid phase. Such
heterogeneous receptors can be used for immunoassays or other
applications where the receptor in an immobilized form can be used
beneficially.
[0057] In a preferred embodiment of the invention the solid phase
is a chromatography carrier material onto which the Fc receptor is
fixed, e.g. sepharose, dextransulfate etc. Such chromatography
materials with Fc receptors bound thereto can beneficially be used
for the adsorption of immunoglobulins from the blood, plasma or
serum of patients or from the culture supernatant of immunoglobulin
producing cells (meaning concentration, enrichment and purification
of antibodies).
[0058] On the one hand, the antibodies bound to the chromatography
material can be eluted and, for example, the immune status of a
patient can thereby be determined. On the other hand, antibodies
from the blood of a patient can thereby be enriched before carrying
out further tests, which is a further preferred embodiment of the
present invention. In many cases it is difficult to conduct
diagnostic assays using blood samples if the latter contains only a
very small number of the antibodies to be identified. By means of a
concentration using a specific chromatographic column with Fc
receptors according to the present invention, antibodies of
interest can easily be concentrated and separated from many other
substances which might disturb the test.
[0059] Basically, it is also possible to use a chromatography
material according to the present invention in an extracorporeal
perfusion system for lavage of the blood in case of certain
diseases where the removal of antibodies plays a crucial role.
[0060] It is, however, also possible to use another material as
solid phase to which the soluble Fc receptor according to the
invention is coupled, e.g. microtiter plates or small reaction
vessels to the walls of which Fc receptors are bound either
directly or indirectly. Such solid phases and vessels can be
particularly important for diagnostic methods, as they enable
screening by using immunoassays e.g. for detecting the presence of
certain immunoglobins in patients' blood or other body fluids.
[0061] To sum up, the recombinant soluble Fc receptors provided by
the present invention as well as the corresponding structure
determination of crystalline preparations of these receptors and of
crystalline complexes of receptors and immunoglobins enable for the
first time to perform a rational drug design, wherefrom it is
possible to modulate the interaction between immunoglobulins and Fc
receptors on cells or soluble receptors. Such a modulation is
preferably an inhibition, whereby the inhibition of the formation
of a complex from IgG and Fc receptor takes place by covering and
preferably by binding of inhibitor molecules to the Fc receptor or
the immunoglobulin. There are various medical applications for such
modulating drugs and in particular of inhibitors and only few of
these applications have been exemplary mentioned within the
framework of the present specification. This can and should by no
means exclude the applicability of such molecules which have been
designed or screened on the basis of the findings about the
molecular structure or FcR/Ig complexes disclosed herein for the
treatment or prevention of other health disturbances.
[0062] The following Examples are to further illustrate the
invention in conjunction with the Figures.
EXAMPLE 1
shFc.gamma.RIIb (Soluble Human Fc.gamma.RIIb)
1.1 Cloning and Expression
[0063] The cDNA of human Fc.gamma.RIIb2 (Engelhardt et al, 1990)
was modified using mutagenous PCR (Dulau et al, 1989). Therefore, a
forward primer was used for the introduction of a new start
methionine after the cleavage site of the signal peptide within a
NcoI site (5'-AAT AGA ATT CCA TGG GGA CAC CTG CAG CTC CC-3') (SEQ
ID NO: 19), while the reverse primer introduced a stop codon
between the putative extracellular part and the transmembrane
region followed by a Sa/I site (5'CCC AGT GTC GAC AGC CTA AAT GAT
CCC C-3') (SEQ ID NO: 20). The PCR product was digested with NcoI
and Sa/I, cloned into a pET11d expression vector (Novagen) and the
proposed sequence was confirmed. The final construct was propagated
in BL21 (DE3) (Grodberg and Dunn, 1988). For the overexpression of
Fc.gamma.RIIb a single colony of the transformed bacteria was
inoculated in 5 ml LB medium containing 100 .mu.g ampicillin per ml
(LB-Amp 100) and incubated overnight at 37.degree. C. The culture
was diluted 200-fold in LB-Amp 100 and incubation was continued
until an OD600 of 0.7-0.9 was achieved. The overproduction of the
protein was induced by adding IPTG to a final concentration of 1
mM. After a growing period of 4 hours the cells were harvested by
centrifugation (30 min, 4000.times.g) and resuspended in
sonification buffer (30 mM sodium phosphate, 300 mM sodium
chloride, 0.02% sodium azide, pH 7.8). After addition of 0.1 mg
lysozyme per ml suspension and incubation for 30 min at room
temperature the sonification was performed on ice (Branson
Sonifier, Danbury, Conn.; Macrotip, 90% output, 80% interval, 15
min). The suspension was centrifuged (30 min, 30,000.times.g) and
resuspended with a Dounce homogenizer in sonification buffer
containing 0.5% LDAO. The centrifugation step and resuspension in
LDAO containing buffer was repeated once before this procedure was
repeated twice without LDAO. The purified inclusion bodies were
stored at 4.degree. C.
1.2 Refolding and Purification of Soluble Human Fc.gamma.RIIb
(shFc.gamma.RIIb)
[0064] The purified inclusion bodies were dissolved to a protein
concentration of 10 mg/ml in 6 M guanidine chloride, 100 mM
2-mercaptoethanol and separated from the insoluble matter by
centrifugation. The refolding was achieved by rapid dilution.
Therefore, one ml of the inclusion body solution was dropped under
stirring within 15 hours into 400 ml of the refolding buffer (0.1 M
TRIS/HCI, 1.4 M arginine, 150 mM sodium chloride, 5 mM GSH, 0.5 mM
GSSG, 0.1 mM PMSF, 0.02% sodium azide, pH 8.5, 4.degree. C.).
Afterwards, the mixture was stirred for 2-3 days until the
concentration of free thiol groups was reduced to 1 mM by air
oxidation as measured according to Ellman (Ellman, 1959). The
solution was dialyzed against PBS and sterile filtered before it
was concentrated 10-fold in a stirring cell equipped with a 3 kD
MWCO ultrafiltration membrane. The protein solution was applied to
a hlgG sepharose column (50 mg hlgG per ml sepharose 4B). Unbound
protein was washed out with 50 mM TRIS pH 8.0 before elution of
Fc.gamma.RIIb by pH jump (150 mM sodium chloride, 100 mM glycine,
0.02% sodium azide, pH 3.0). The eluate was immediately neutralized
with 1 M TRIS pH 8.0. The Fc.gamma.RIIb containing solution was
concentrated and subjected to gel filtration on a Superdex-75
column equilibrated with crystallization buffer (2 mM MOPS 150 mM
sodium chloride, 0.02% sodium azide pH 7.0). The fractions
containing Fc.gamma.RIIb were pooled, concentrated to 7 mg/ml and
stored at -20.degree. C.
1.3 Equilibrium Gel Filtration Experiments
[0065] A Superdex75 column was connected to FPLC and equilibrated
with PBS containing 10 .mu.g shFcRIIb per ml. Human Fc fragment was
solved to a concentration of 1 .mu.g/10 .mu.l in the equilibration
buffer and injected. The resulting chromatogram yielded a positive
peak comprising the complex of the shFc.gamma.RIIb and the Fc
fragment while the negative peak represents the lack of receptor
consumed from the running buffer for complex formation.
1.4 Crystallization and Data Collection
[0066] Initial crystallization trials employing a 96 condition
sparse matrix screen (Jancarik and Kim, 1991) were performed in
sitting drops at 20.degree. C. using the vapor diffusion method.
Occurring. crystals were improved by changing the pH as well as the
salt, precipitant and additive concentration. Diffraction data from
suitable crystals was collected on an image plate system (MAR
research) using graphite monochromated CuK.sub.a radiation from a
RU200b rotating anode generator (Rigaku) operated at 50 kV and 100
mA. The reflections were integrated with the program MOSFLM
(Leslie, 1997) and subsequently the data was scaled, reduced and
truncated to obtain the structure-factor amplitudes using routines
from the CCP4 program suite (Collaborative Computational Project,
1994).
1.5 Summary of Expression, Purification and Refolding of
shFc.gamma.RIIb
[0067] The extracellular part of Fc.gamma.RIIb was expressed in
high levels under the control of a T7 promoter in the T7 RNA
polymerase positive E. coli strand BL21/DE3 (Grodberg & Dunn,
1988). The protein was deposited in inclusion bodies, which were
employed in the first purification step. The isolation of the
inclusion bodies was started with an intense combined
Iysozyme/sonification procedure to open virtually all cells which
would otherwise contaminate the product. The subsequent washing
steps with the detergent LDAO, which has excellent properties in
solving impurities but not the inclusion bodies itself already
yielded a product with a purity of >90% (FIG. 1).
[0068] This product was used for refolding trials without further
purification. The inclusion bodies were dissolved in high
concentration of 2-mercaptoethanol and guanidine to ensure the
shift of covalent and non-covalent aggregates to monomers. This
solution was rapidly diluted with refolding buffer to minimize
contacts between the unfolded protein molecules which would
otherwise form aggregates. The use of arginine in the refolding
buffer prevents the irreversible modification of side chains as
often recognized with urea. After addition of the protein to the
refolding buffer, the solution was stirred at 4.degree. C. until
the concentration of free thiol groups was reduced to 1 mM, which
was absolutely necessary as earlier dialysis resulted in
an.cndot.inactive product. In a second purification step the
dialyzed and refolded Fc.gamma.RIIb was bound to immobilized hlgG
to remove minor fractions of E. coli proteins and inactive
receptor. The protein was eluted with a pH jump and immediately
neutralized. After this affinity chromatography step
shFc.gamma.RIIb is essentially pure except for a minor
contamination resulting from the coeluting IgG which leached out of
the matrix even after repeated use (FIG. 1). The IgG as well as
receptor multimers which are not visible in the reducing SDS-PAGE
could easily be removed by gel filtration. Parallel to the removal
of the contaminants in this step the buffer is quantitatively
exchanged. This procedure ensures a defined composition of the
protein solution as even slight variations can cause
irreproducibility of the crystallization attempts or even inhibit
the formation of crystals. Overall 6 mg pure protein could be
gained per litre E. coli culture, which is about 10% from the
Fc.gamma.RIIb content of the inclusion bodies.
[0069] N-terminal protein sequencing revealed the identity with the
expected sequence H.sub.2N-GTPAAP without detectable contamination.
ESI-MS analysis showed that the final material used in
crystallization trials is homogenous with respect to size. From the
primary sequence the molecular weight was calculated to 20434 Da,
which corresponds to 20429 Da found by mass spectroscopy. The
discrepancy lies within the error of the instrument, and no
additional peak for a species containing the leading methionine is
found.
[0070] The crystallization of shFc.gamma.RIIb was performed in
sitting drops using the vapor diffusion method. Initial trials with
a sparse matrix screen (Jancarik & Kim, 1991) resulted already
in small crystalline needles. Subsequent optimization of the
preliminary crystallization condition by varying precipitant, salt,
their concentration and pH led to the isolation of three different
crystal forms. Orthorhombic crystals grew from mixture of 1.5 .mu.l
reservoir solution (33% PEG2000, 0.2 M sodium acetate, pH 5.4) with
3 .mu.l of the protein solution. They appeared within 3 days and
reached their final size of approximately 80 .mu.m.times.80
.mu.m.times.500 .mu.m after one week. These crystals diffracted to
1.7 .ANG.. Crystals could also be grown in two other space groups
from reservoir solution containing 26% PEG8000, 0.2 M sodium
acetate, pH 5.6, 5 mM Zn(OAc).sub.2 100 mM sodium chloride
(hexagonal form) and 26% PEG8000, 0.2 M NaOAc, pH 5.6, 10% (v/v)
1,4-Dioxan, 100 mM sodium chloride (tetragonal form). These
crystals were of suitable size for X-ray analysis but diffracted
only to 2.7 .ANG. and 3.8 .ANG. for the tetragonal and hexagonal
crystal form respectively (Table 1).
[0071] Fc.gamma.RII was expressed in E. coli which, besides the
comparatively low production costs and the availability, has
several advantages especially when the glycosylation performed by
mammalian cells is not necessary for the function of the protein as
in the case of Fc.gamma.RII where IgG binding occurs independently
of carbohydrate attachment (Sondermann et al, 1998A). In E. coli a
homogenous product can reproducibly be generated, which is in
contrast to the expression in mammalian cells where batch dependent
variances are often observed. In such a system the product is for
several days exposed to proteases at temperatures of more than
30.degree. C. In contrary, the expression of the protein in E. coli
under the control of the strong T7 promoter at 37.degree. C.
frequently leads to the formation of protease inaccessible
inclusion bodies. A further advantage of the expression in bacteria
is that the material could be considered to be free of pathogenic
germs, which might derive from employed fetal calf serum or the
cell line itself. In mammalian expression particular care must be
taken during the purification of the target protein because
potential effective hormones or growth factors might be copurified.
One case where the effects of sFc.gamma.R were ascribed to a
TGF.beta.1 contamination is already reported (Galon et al,
1995).
1.6 Purification
[0072] The purification procedure is straightforward. It consists
of three steps which can easily be performed in a single day. The
protein is obtained in a pure form and in high yields and could
even be obtained in considerable quality without the expensive IgG
affinity column. The success of such a protocol would depend on the
careful preparation of the inclusion bodies, as most of the
impurities can be eliminated already in the first purification
step.
1.7 Characterization
[0073] The purified Fc.gamma.RIIb was characterized by SDS-PAGE and
isoelectric focussing as well as N-terminal sequencing and mass
spectroscopy. Thus, the material can be considered pure and
homogeneous with respect to its chemical composition, but the
intriguing question whether the receptor is correctly folded
remains to be discussed. All cysteins are paired, since no free
thiol groups are detected with Ellman's test. The material is
monomeric and eludes with the expected retention time in peaks of
symmetrical shape from a size exclusion chromatography column.
Furthermore, Fc.gamma.RIIb binds to IgG sepharose, recombinant
Fc.gamma.RIIb from E. coli is active because it specifically binds
IgG.
1.8 Crystallization
[0074] The orthorhombic crystal form of Fc.gamma.RIIb diffracted
X-rays to a resolution of 1.7 .ANG., which is a drastic improvement
compared to previously reported crystals of the same molecule
derived from insect cell expression (Sondermann et al, 1998A).
These crystals diffracted to 2.9 .ANG. and were of space group
P3.sub.121. Thus, the glycosylation of the insect cell derived
receptor influences the crystallization conditions. Instead of the
trigonal space group, three different crystal forms are found.
After a possible solution of the structure these crystal forms will
help identify artificial conformations of the protein due to
crystal contacts.
[0075] Fc.gamma.Rs do not exhibit any sequence similarity to other
proteins but due to a conserved cystein spacing they are affiliated
to the immunoglobulin super family. Consequently, we tried to solve
its structure by molecular replacement, but extensive trials using
IgG domains from a variety of molecules failed. Thus the structure
of Fc.gamma.RIIb has to be solved by the methods of multiple
isomorphous replacement.
[0076] We have shown for the first time that Fc.gamma.RIIb can be
obtained in an active form from E. coli. This is the basis for
crystallographic investigations that will soon, due to the already
gained crystals of exceptional quality, result in the structure
solution of this important molecule. The structure will provide
information on the IgG binding site and provide a starting point
for the knowledge based design of drugs that interfere with
recognition of the ligand by its receptor. Furthermore, because of
the high homology between Fc.gamma.RIIb and other FcRs including
Fc.epsilon.RIa it seems possible that these molecules can be
produced in the same way, which would provide valuable material for
the ongoing research.
1.9 Methods
Protein Chemistry
[0077] Recombinant soluble human Fc.gamma.RIIb was expressed in E.
coli, refolded purified and crystallized as described elsewhere
(Sondermann et al, 19988). Briefly, the putative extracellular
region of hFc.gamma.RIIb2 (Engelhardt et al, 1990) was
overexpressed in E. coli. Inclusion bodies were purified by
lysozyme treatment of the cells and subsequent sonification. The
resulting suspension was centrifuged (30 min 30,000.times.g) and
washed with buffer containing 0.5% LDAO. A centrifugation step and
resuspension in LDAO containing buffer was repeated once before
this procedure was repeated twice without LDAO. The inclusion
bodies were solved in 6 M guanidine hydrochloride and the protein
was renaturated as described. The dialyzed and filtrated protein
solution was applied to a hlgG sepharose column and eluted by pH
jump. The concentrated neutralized fractions were subjected to
size-exclusion chromatography on a Superdex-75 column (26/60,
Pharmacia).
Crystallization
[0078] Crystallization was performed in sitting drops at 20.degree.
C. using the vapor diffusion technique. Crystallization screens
were performed by changing pH, salt, precipitant and additives. The
final crystals used for data collection were grown in 33% PEG2000,
0.2 M sodium acetate, pH 5.4 (orthorhombic form) 26% PEG8000, 0.2 M
sodium acetate, pH 5.6, 10% (v/v) 1,4-dioxane, 100 mM sodium
chloride (tetragonal form), and 26% PEG8000, 0.2 M sodium acetate,
pH 5.6, 5 mM ZN(OAc).sub.2, 100 mM sodium chloride (hexagonal
form). The insect cell derived protein was crystallized in 32%
PEG6000, 0.2 M sodium, pH 5.3.
Preparation of Heavy-Atom Derivatives
[0079] The heavy-atom derivatives were prepared by soaking the
crystals in the crystallization buffer containing 2 mM
platinum(II)-(2,2'-6,2''terpyridinium) chloride for 24 hours or 10
mM uranylchloride for 8 days.
X-Ray Data Collection
[0080] Diffraction data was collected on an image plate system (MAR
research) using graphite monochromated CuK.sub.a radiation from a
RU200b rotating anode generator (Rigaku) operated at 50 kV and 100
mA. The reflections were integrated with the program MOSFLM 5.50
(Leslie, 1997) and subsequently the data was scaled and truncated
to obtain the structure-factor amplitudes using routines from the
CCP4 program suite (Collaborative Computational Project, 1994).
Structure Determination
[0081] The structure was solved with the standard procedures of the
MIR method. From the large-number of soaks carried out with
different heavy-atom components only the two compounds yielded
interpretable Patterson maps. The heavy-atom positions for each
derivative were determined from difference Patterson maps and
initial phases were calculated. Cross-phased difference Fourier
maps were used to confirm heavy atom positions and establish a
common origin for the derivatives. Anomalous data were included to
discriminate between the enantiomers. The heavy atom parameters
were further refined with the program MLPHARE from the CCP4 package
leading to the statistics compiled in Table 2. An electron-density
map was calculated to a resolution of 2.1 .ANG. and the phases were
improved further by solvent flattening and histogram matching with
the program DM from the CCP4 suite. The resulting electron density
map was of sufficient quality to build most of the amino acid
residues. Model building was performed with 0 (Jones et al, 1991)
on an Indigo2 work station (Silicon Graphics Incorporation). The
structure refinement was done with XPLOR (Brunger et al, 1987) by
gradually increasing the resolution to 1.7 .ANG. using the
parameter set of Engh and Huber (Engh & Huber, 1991). When the
structure was complete after several rounds of model building and
individual restraint B-factors refinement
(R.sub.fac=29%/R.sub.free=36%), 150 water molecules were built into
the electron density when a Fo-Fc map contoured at 3.5 .sigma.
coincided with well defined electron density of a 2Fo-Fc map
contoured at 1.sigma.. The resulting refinement statistic is shown
in Table 3.
1.10 Structure Determination
[0082] The crystal structure of recombinant soluble human
Fc.gamma.RIIb was solved by multiple isomorphous replacement (MIR)
to 1.7 .ANG. resolution, since a structure solution by molecular
replacement with isolated domains of the Fc fragment from human
IgG1 (Huber et al, 1976, PDB entry 1fc1; Deisenhofer, 1981) failed.
The putative extracellular part of the receptor (amino acid
residues 1-187 as depicted in SEQ ID NO:2) was used for
crystallization trials (Sondermann et al, 1998B) while the model
contains the residues 5-176 as the termini are flexible and not
traceable into the electron density. Additionally, the model
contains 150 water molecules and the refinement statistics are
summarized in Table 2. The structure contains a cis proline at
position 11. None of the main chain torsion angles is located in
disallowed regions of the Ramachandran plot. The fully refined
model was used to solve the structure of the same protein in
crystals of space group P4.sub.22.sub.12 and of the glycosylated
form derived from insect cells in crystals of space group
P3.sub.121 (Table 2).
[0083] The polypeptide chain of Fc.gamma.RIIb folds into two
Ig-like domains as expected from its affiliation with the
immunoglobulin super family. Each domain consists of two beta
sheets that are arranged in a sandwich with the.cndot.conserved
disulfide bridge connecting strands B and F on the opposing sheets
(FIG. 3). Three anti-parallel .beta.-strands (A1, B, E) oppose a
sheet of 5 .beta.-strands (C', C, F, G, A21, whereby strand A1
leaves the 3-stranded, B-sheet and crosses over to the 4-stranded
anti-parallel sheet adding the short parallel 5th strand A2. The
arrangement of secondary structure elements as well as their
connectivity is identical in both domains of the Fc.gamma.RIIb and
a rigid body fit of one domain onto the other revealed a r.m.s.
distance of 1.29 .ANG. of 67 matching C.alpha. atoms.
[0084] The domains are arranged nearly perpendicularly to each
other enclosing an angle of 70 degrees between their long axes
forming a heart-shaped overall structure. This arrangement results
in an extensive contact region between the domains (FIG. 4).
Residues from strand A2 and from the segment linking A2 and A1 of
the N-terminal domain intermesh with residues of strands A1 and B
from the C-terminal domain. This region is tightly packed and the
interaction is strengthened by several hydrogen bonds resulting in
a rigid arrangement. This is confirmed by the conservation of the
structure in three different space groups. In orthorhombic,
tetragonal and hexagonal (insect cell derived) crystal forms a
deviation of less than 2.degree. in the interdomain angle is
found.
1.11 Overall Structures
[0085] The structure of recombinant human Fc.gamma.RIIb derived
from E. coli was solved by MIR to 1.7 .ANG. resolution from
orthorhombic crystals. An essentially identical structure is found
in tetragonal and with protein derived from insect cells in
hexagonal crystals. In all three structures the last nine residues
of the polypeptide chain were found disordered. The flexibility of
the C-terminal linker region between the structured core of the
molecule and the transmembrane part may be functionally relevant to
allow some reorientation of the receptor to enhance the recognition
of the Fc parts in immunocomplexes.
1.12 Homologue Receptors
[0086] The Ig domains found in the Ig super family of proteins are
characterized by a beta sandwich structure with a conserved
disulfide bridge connecting two strands of the opposing sheets. The
typical arrangement of 3 and 4 anti parallel beta strands that form
a sandwich as found in Fc.gamma.RIIb occurs also in the T cell
receptor, Fc fragment, CD4 or the Fab fragment. A structural
alignment of the individual Ig domains of these molecules with the
two domains of Fc.gamma.RIIb shows a common, closely related
structure. The relative arrangement of the domains, however, is not
related in these molecules and covers a broad sector. Despite the
structural similarity between Ig domains from different molecules
and the strikingly low r. m. s. deviation of C.alpha. atoms that
result when the two domains of Fc.gamma.RII are superimposed, no
significant sequence similarity is found (FIGS. 5a and 5b). A
structure-based sequence alignment shows a conserved hydrophobicity
pattern along the sequence of the domains, together with, beside
the cysteins, only few identical amino acid residues. We first
prepared a structure-based alignment of the two C-terminal domains
of the IgG1 heavy chain and the Fc.gamma.RIIb and added the
sequences of the other related Fc.gamma.R and the Fc.gamma.RIa
domains. This shows that the sequences of the three domain
Fc.gamma.RI and the two domain receptors are compatible with the
hydrophobicity pattern of Ig domains and several conserved amino
acid residues are revealed. Firstly, the different domains of an
FcR are more related to each other than to Ig domains from other
molecules of the Ig super family. Secondly, the N-terminal domains
of the receptors relate to each other as the second domains do.
Thirdly, the sequence of the third domain of Fc.gamma.RI shows
features from both groups of domains. Taken together, we confirm
the affiliation of the FcRs to the Ig super family and speculate
that all FcR-domains originate from a common ancestor, an ancient
one domain receptor that acquired a second domain by gene
duplication. Further divergent development of such a two domain
receptor resulted in the present diversity, including Fc.gamma.RI
that acquired a third domain.
[0087] Conservation of these amino acid residues that contribute to
the interdomain contact in Fc.gamma.RIIb in the alignment are a
hint to a similar domain arrangement in different receptors. In
Table 4 the residues contributing with their side chains to the
interdomain contact (FIG. 4) are compiled for Fc.gamma.RIIb
together with the corresponding amino acid residues in other
receptors according to the structure-based sequence alignment of
FIG. 5b. Except for Asn 15, which is not conserved between the
FcRs, the involved residues are identical or conservatively
replaced providing strong support for a similar structure and
domain arrangement in all FcRs.
1.13 The Contact Interface to IgG
[0088] Limited information about the interactions of FcRs with
their ligands is available from mutagenesis studies (Hogarth et al,
1992; Hulett et al, 1994; Hulett et al, 1995). By systematically
exchanging loops between the .beta.-strands of Fc.gamma.RIIa for
Fc.epsilon.RIa amino acid residues the B/C, C'/E and F/G loops of
the C-terminal domain were evaluated as important for ligand
binding (FIG. 3, FIG. 5b). In the structure model these loops are
adjacent and freely accessible to the potential ligand.
Additionally, most of the amino acid residues in these loops were
exchanged for alanines by single site mutations which resulted in a
drastic alteration of the affinity of Fc.gamma.RIIa to dimeric
human IgG1. Also, the single amino acid exchange Arg 131 to His in
the C-terminal domain (C'/E loop) in the high responder/low
responder polymorphism, which alters the affinity of the
Fc.gamma.RIIa to murine IgG1, points to that region. Thus, the
amino acid residues in this area are either important for ligand
binding or the structural integrity of that region. Here, the
structure shows a clustering of the hydrophobic amino acid residues
Pro 114, Leu 115 and Val 116 in the neighbourhood of Tyr 157. This
patch is separated from the region Leu 159, Phe 121 and Phe 129 by
the positively charged amino acid residues Arg 131 and Lys 117
which protrude from the core structure (FIG. 5b).
1.14 Glycosylation
[0089] In the sequence of Fc.gamma.RIIb three potential
N-glycosylation sites are found. All three sites are on the surface
of the molecule and are accessible. They are located in the E/F
loops (N61 and N142) of both domains and on strand E (N135) of the
C-terminal domain (FIG. 3, FIG. 6). Since the material used for the
solution of this structure was obtained from E. coli, it does not
contain carbohydrates, while the FcRs isolated from mammalian cells
are highly glycosylated. The three potential glycosylation sites
are located rather far from the putative IgG binding region, and
non-glycosylated Fc.gamma.RIIb binds human IgG, suggesting a minor
role of glycosylation in binding. This was confirmed by the
structure of the Fc.gamma.RIIb produced in insect cells which is
glycosylated (Sondermann et al, 1998A). Except for a 2.degree.
change of the interdomain angle possibly due to different crystal
contacts, no differences between the glycosylated and
unglycosylated protein structures were found. The three
glycosylation sites are only optionally used as shown by SDS-PAGE
where the material appears in 4 bands. No additional electron
density for those sugars was found a consequence of chemical and
structural heterogeneity.
EXAMPLE 2
shFc.gamma.RIIa (Soluble Human Fc.gamma.RIIa)
[0090] The procedures were performed according to example 1 except
for the indicated changes:
2.1 Cloning and Expression
[0091] shFc.gamma.RIIa was generated by mutating the respective
wild-type cDNA (Stengelin et al., 1988) and expressed according to
example 1 with the mutagenous primers listed in table 5. For the
expression of the protein a pET22b+vector was chosen.
2.2 Refolding and Purification
[0092] shFc.gamma.RIIa was refolded according to example 1 with the
respective refolding buffer listed in table 6.
2.3 Crystallisation
[0093] shFc.gamma.RIIa was crystallised as described under
conditions indicated in table 7.
2.4 Structure Determination
[0094] The structure was solved with the method of isomorphous
replacement with shFc.gamma.RIIb as search model.
EXAMPLE 3
shFc.gamma.RIII (Soluble Human Fc.gamma.RIII)
[0095] The procedure was performed according to example 1 except
for the indicated changes:
3.1 Cloning and Expression
[0096] shFc.gamma.RIII was generated by mutating the respective
wild-type cDNA (Simmons & Seed, 1988) and expressed according
to example 1 with the mutagenous primers listed in table 5. For the
expression of the protein a pET22b+vector was chosen.
3.2 Refolding and Purification
[0097] shFc.gamma.RIII was refolded according to example 1 with the
respective refolding buffer listed in table 6.
3.3 Crystallisation
[0098] shFc.gamma.RIII was crystallised as described under
conditions indicated in table 7.
3.4 Structure Determination
[0099] The structure was solved with the method of isomorphous
replacement with shFc.gamma.RIIb as search model.
3.5 Crystallisation of a shFc.gamma.RIII:hFc1 Complex
[0100] hlgG1 derived from the serum of a myeloma patient was used
to prepare Fc-fragments (hFc1) by digestion with plasmin
(Deisenhofer et al., 1976). The resulting Fc-fragments were
separated from the Fab-fragments by protein A chromatography.
Partially digested hlgG was removed by size exclusion
chromatography with MBS (2 mM MOPS, 150 mM NaCl, 0.02% sodium
azide, pH 7.0) as running buffer. Equimolar amounts of hFc1 and
shFcgRIII were mixed and diluted with MBS to a concentration of 10
mg/ml. The complex was crystallised as described under conditions
indicated in table 5.
EXAMPLE 4
shFc.epsilon.RII (Soluble Human Fc.epsilon.RII)
[0101] The procedure was performed according to example 1 except
for the indicated changes:
4.1 Cloning and Expression
[0102] Fc.epsilon.RII was generated by mutating the respective
wild-type cDNA (Kikutani et al., 1986) and expressed according to
example 2 with the mutagenous primers listed in table 5. For the
expression of the protein a pET23a+vector was chosen.
4.2 Refolding and Purification
[0103] Refolding of shFc.epsilon.RII was achieved as described in
example 1, with the exception that prior to rapid dilution the
dissolved inclusion bodies were dialysed against 6M guanidine
chloride, 20 mM sodium acetate, pH 4.0. shFc.epsilon.RII was
refolded according to example 1 with the respective refolding
buffer listed in table 6. After refolding the protein solution was
dialysed against PBS, concentrated 100-fold and purified by gel
filtration chromatography on Superdex 75. This yielded pure
shFc.epsilon.RII which was dialysed against 2 mM TRIS/. HCl, 150 mM
NaCl, 0.02% sodium azide, pH 8.0, concentrated to 10 mg/ml and
stored at 4.degree. C.
EXAMPLE 5
shFc.gamma.RI (Soluble Human Fc.gamma.RI)
[0104] The procedure was performed according to example 1 except
for the indicated changes:
5.1 Cloning and Expression
[0105] shFc.gamma.RI was generated by mutating the respective
wild-type cDNA (Allen & Seed, 1988) and expressed according to
example 1 with the mutagenous primers listed in table 5. For the
expression of the protein a pET32a+vector was chosen, which
contains after the N-terminal thioredoxin a hexahistidine-tag with
a C-terminal thrombin cleavage site followed by the shFc.gamma.RI
in frame with the mentioned proteins and amino acid residues. For
the overexpression of the fusion protein the E. coli strain BL21
(DE3) containing the plasmids pUBS and pLysS (Novagen) was
used.
[0106] The purified inclusion bodies were solubilised in 6M
guanidine-HCI, 10 mM .beta.-mercaptoethanol, 50 mM Tris pH8.0 and
bound to a Ni-NTA column (Qiagenl. The elution was performed with
an imidazole gradient ranging from 0 to 1M imidazole. The eluted
protein was dialysed against a 1000 fold volume of 150 mM NaCl, 50
mM Tris pH8.0, 2 mM GSH, 0.5 mM GSSG for 24 hours at 4.degree. C.
After concentrating the protein solution to 25% of the initial
volume, thrombin was added. After 6 h of incubation at 37.degree.
C. the N-terminal thioredoxin and the His-tag were removed
completely as verified by N-terminal sequencing. During this
digestion the shFcgRI precipitated quantitatively out of
solution.
5.2 Refolding and Purification
[0107] shFc.gamma.RI was refolded according to example 1 with the
respective refolding buffer listed in table 6. After the redox
potential decreased to 1 mM the solution was dialysed against PBS
pH8.0 and concentrated.
[0108] The refolded Protein was analysed by size exclusion
chromatography, which yielded a peak of the proposed monomeric
receptor and non reducing SDS-PAGE which showed a major band at 30
kDa.
EXAMPLE 6
shFc.epsilon.RIa (Soluble Human Fc.epsilon.RIa)
[0109] The procedure was performed according to example 1 except
for the indicated changes:
6.1 Cloning and Expression
[0110] shFc.epsilon.RI was generated by mutating the respective
wild-type cDNA (Kochan et al., 1988) and expressed according to
example 1 with the mutagenous primers listed in table 5. For the
expression of the protein a pET23a+vector was chosen.
BRIEF DESCRIPTION OF THE FIGURES
[0111] FIG. 1: 15% reducing SDS PAGE showing the purification of
sFc.gamma.RIIb
[0112] Lane 1: Molecular weight marker. Lane 2: E. coli lysate
before induction. Lane 3: E. coli lysate 1 h after induction. Lane
4: E. coli lysate 4 h after induction. Lane 5: Purified inclusion
bodies of sFc.gamma.RIIb. Lane 6: Eluate of the hlgG affinity
column. Lane 7: Pooled fractions of the gel filtration column.
[0113] FIG. 2: Equilibrium gel filtration
[0114] 1 .mu.g hFc solved in 10 .mu.l equilibration buffer (10
.mu.g sFc.gamma.RIIb/ml PBS) was applied to a size exclusion
chromatography column and the absorbance of the effluent was
measured (280 nm) as a function of time. The injected Fc fragment
forms a complex with the sFc.gamma.RIIb in the equilibration buffer
(t=22 min). The negative peak of consumed sFc.gamma.RIIb is
observed at t=26 min.
[0115] FIG. 3: Overall structure of human sFc.gamma.RIIb
[0116] Stereo ribbon representation of the sFc.gamma.RIIb
structure. The loops supposed to be important for IgG binding are
depicted in red with some of the residues within the binding site
and the conserved disulfide bridge in ball and stick
representation. The potential N-glycosylation sites are shown as
green balls. The termini are labeled and the .beta.-strands are
numbered consecutively for the N-terminal domain in black and for
the C-terminal domain in blue. The figure was created using the
programs MOLSCRIPT (Kraulis, 1991) and RENDER (Merritt and Murphy,
1994).
[0117] FIG. 4: Interdomain contacts
[0118] The figure shows a close-up on the residues involved in the
interdomain contacts of sFc.gamma.RIIb. The amino acid residues of
the N-terminal domain are depicted blue and the residues of the
C-terminal domain yellow. The model is covered by a 2Fo-Fc electron
density contoured at 1.sigma. obtained from the final coordinates.
Hydrogen bridges between the domains are represented by white
lines. The figure was created using the program MAIN (Turk,
1992).
[0119] FIG. 5a: Superposition of the two Fc.gamma.RIIb domains and
the CH2 domain of human IgG1
[0120] Both domains of Fc.gamma.RIIb and the CH2 domain of hlgGl
were superimposed. The N-terminal domain is depicted in blue, the
C-terminal domain in red and the CH2 domain of hlgGl in green. The
respective termini are labeled and the conserved disulfide bridges
are depicted as thin lines.
[0121] FIG. 5b: Structure based sequence alignment of the
sFc.gamma.FIIb domains with domains of other members of the FcR
family
[0122] The upper part of the figure shows the structure based
sequence alignment of the Fc.gamma.RIIb and hlgGl Fc fragment
domains performed with the program GBF-3D-FIT (Lessel &
Schomburg, 1994). Amino acid residues with a C.alpha. distance of
less than 2.0 .ANG. in the superimposed domains are masked: lilac
for matching residues between the Fc fragment domains; yellow for
residues in the Fc.gamma.RIIb domains; and green when they can be
superimposed in all four domains. The .beta.-strands are indicated
below this part of the alignment and are labeled consistent with
FIG. 3.
[0123] The lower part of the figure shows the alignment of the
amino acid sequences from the other Fc.gamma.Rs and the homologue
FC.epsilon.RIa to the profile given in the upper part of the figure
using routines from the GCG package (Genetics Computer Group,
1994). The upper and lower row of numbering refer to the N- and
C-terminal domains of Fc.gamma.RIIb. The conserved cysteins are
typed in magenta and the potential glycosylation sites in blue.
Identical residues within the first domain are masked orange, those
in the second domain pink and green when the residues are conserved
within both domains. The less conserved third domain of Fc.gamma.RI
is aligned between the first and the second domains. Red arrows
point to residues that are involved in side chain contacts between
the first and the second domain while blue arrows depict residues
that are relevant for IgG binding. The figure was produced with the
program ALSCRIPT (Barton, 1993).
[0124] FIG. 6: The putative binding sites of Fc.gamma.RIIb
[0125] Solid surface representations of Fc.gamma.RIIb as produced
with GRASP (Nicholls et al, 1991), the color coding is according to
the relative surface potential from negative (red) to positive
(blue). FIG. 6a shows the molecule as in FIG. 3 by a rotation of
about 90.degree. counter-clockwise around the vertical. In FIG. 6b
the molecule is rotated 90.degree. clockwise around the same axis.
Both views show the putative binding regions on the C-terminal
(FIG. 6a) and the N-terminal domain (FIG. 6b). The amino acid
residues discussed in the text are labeled.
[0126] FIG. 7: C.alpha.-trace of the superpositioned structures of
the Fc.gamma.-receptors
[0127] Fc.gamma.RIII red, Fc.gamma.RIIa green and Fc.gamma.RIIb
blue. Residues important for IgG binding are shown in
ball-and-stick. The N- and C-termini are labelled.
[0128] FIG. 8: Overview of the Fc.gamma.RIII/Fc-fragment crystal
structure in ribbon representation
[0129] The sugar residues bound to the Fc-Fragment are indicated in
ball-and-stick. The Fc.gamma.RIII (blue) binds in the lower hinge
region between chain-B (red) and chain-A (green) of the
Fc-fragment.
[0130] FIG. 9: Close-up on the binding region of the Fc.gamma.RIII
and the Fc-fragment
[0131] The colour scheme is in agreement to FIG. 8 and residues
important for complex formation are shown in ball-and-stick.
[0132] FIG. 10a:
[0133] In the upper part of FIG. 10a a structure based sequence
alignment of the Fc-Receptor ecto-domains is shown. Conserved
residues are shaded yellow and identical residues orange. The lower
part of the figure shows a part of the alignment of human antibody
sequences. Residues 9f the human Fc.gamma.RIII in contact with the
Fc-fragment in the complex crystal structure are connected by lines
(black for hydrophobic interaction, red for salt bridges and blue
for hydrogenbridges. Residues from the Fc-receptor in contact with
the A-chain of the Fc-fragment are connected with dashed lines and
those in contact with the .beta.-chain of the Fc-fragment with
solid lines. Red, blue and black lines represent charged, polar and
other contacts, respectively.
[0134] FIG. 10b:
[0135] In the upper part of FIG. 10b a structure based sequence
alignment of the Fc-Receptor ecto-domains is shown. Conserved
residues are shaded yellow and identical residues orange. Conserved
residues within the less related Kir and FcA-Receptor sequences are
shaded blue. The lower part of the figure shows a part of the
alignment of human antibodies with the mouse IgE (mlgE) sequence.
Residues of the human Fc.gamma.RIII in contact with the Fc-fragment
in the complex crystal structure are connected by lines (black for
hydrophobic interaction, red for salt bridges and blue for
hydrogenbonds). Residues from the Fc-receptor in contact with the
A-chain of the Fc-fragment are connected with dashed lines and
those in contact with the B-chain of the Fc-fragment with solid
lines. Red, blue and black lines represent charged, polar and other
contacts, respectively.
[0136] FIG. 11 and FIG. 12:
[0137] FIG. 11 and FIG. 12 show an alignment of the produced
sFc.gamma.R, sFc.epsilon.RIa and the short form of sFc.epsilon.RII
and the produced sFc.gamma.R and sFc.epsilon.RIa without
sFc.epsilon.RII, respectively.
TABLE-US-00001 TABLE 1 Crystallographic results Orthorhombic
Tetragonal Hexagonal Space group P2.sub.12.sub.12.sub.1 [19]
P4.sub.22.sub.12 [94] P3 [143] Unit cell a = 40.8 .ANG., b = 50.9
.ANG., a = 85.7 .ANG., b = 85.7 .ANG., a = 80.9 .ANG., b = 80.9
.ANG., dimensions c = 80.5 .ANG., .alpha. = 90.degree., c = 63.4
.ANG., .alpha. = 90.degree., c = 157.0 .ANG., .alpha. = 90.degree.,
.beta. = 90.degree., .gamma. = 90.degree. .beta. = 90.degree.,
.gamma. = 90.degree. .beta. = 90.degree., .gamma. = 90.degree.
R.sub.merge 5.8% 9.8% 13.6% Resolution 1.7 .ANG. 2.7 .ANG. 3.8
.ANG. Unique 18,040 6,616 7,210 Completeness 89.1% 97.1% 63.0%
Multiplicity 3.5 4.4 1.3 V.sub.m, molecules 2.09 .ANG..sup.3/Da, 1
mol., 2.91 .ANG./Da, 1 mol, 2.97 .ANG./Da, 5 mol, per asymmetric
41% solvent 58% solvent 59% solvent unit, solved content The
obtained preliminary crystallographic data are shown in this
table.
TABLE-US-00002 TABLE 2 Data collection statistics Completeness No.
of (overall/ No. Space unique Resolution last shell) R.sub.m of
Phasing Derivative Group reflections Multiplicity (.ANG.) (%/%) (%)
sites power NATI P2.sub.12.sub.12.sub.1 18009 3.6 1.74 92.9/86.4
5.5 NATI P4.sub.22.sub.12 6615 4.5 2.70 97.1/94.3 10.4 NATI-
P3.sub.121 3545 2.5 3.0 93.0/98.9 14.4 Baculo UOAc
P2.sub.12.sub.12.sub.1 7722 4.2 2.1 96.8/95.7 7.3 1 1.79 PtP.gamma.
P2.sub.12.sub.12.sub.1 5520 3.9 2.3 89.7/49.6 10.5 1 1.39 R.sub.m =
.SIGMA.1/.sub.h - </.sub.h>1/.SIGMA.</.sub.h> Phasing
power: <F.sub.H>/E; where <F.sub.H> = .SIGMA.
(F.sub.H.sup.2/n).sup.1/2 is the r.m.s. heavy atom structure
amplitude.
[0138] E=.SIGMA.[(F.sub.PHC.sup.-F.sub.PH).sup.2/n].sup.1/2 is the
residual lack of closure error with F.sub.PH being the structure
factor amplitude and F.sub.PHC=IF.sub.P+F.sub.HI the calculated
structure factor amplitude of the derivative.
TABLE-US-00003 TABLE 3 Refinement statistics Resolution range
(.ANG.) 8.0-1.74 .ANG. No. of unique reflections 16252 (F >
0.sigma.(F)) R factor 19.4 R.sub.free* 27.9 No. of atoms per
asymmetric unit protein 1371 solvent 150 Rms deviation from ideal
geometry bond length (.ANG.) 0.009 bond angle (.degree.) 2.007
Average B factor (.ANG..sup.2) protein main chain 18.8 protein side
chain 25.2 solvent 36.7 Rms deviation of bonded B factors 4.1
(.ANG..sup.2) *R.sub.free: 5% of the reflections were used as a
reference data set and were not included in the refinement.
TABLE-US-00004 TABLE 4 Residues that contribute to the interdomain
contact via side chains Fc.gamma.RIIb Fc.gamma.RIIa Fc.gamma.RIII
Fc.gamma.RI Fc.epsilon.RIa Asn15 Asn Ser Ser Arg Asp20 Asp Asp Glu
Glu Gln91 Gln Gln Gln Gln His108 His His His His Trp110 Trp Trp Trp
Trp
TABLE-US-00005 TABLE 5 Primers used for the amplification of the
FcRs Construct 5'-Prmer 3'-Primer sFc.gamma.RI
5--CACCCATATGGCAGTGATCTCTTT-3' 5'-AGGACTCGAGACTAGACAGGAGTTGGTAAC-3'
sFc.gamma.RIIa 5'-ACAGTCATATGGCAGCTCCCC-3'
5'-AAAAAAAGCTTCAGGGCACTTGGAC-3' sFc.gamma.RIIb
5'-AATTCCATGGGGACACCTGCAGCTCCC-3'
5'-CCCAGTGTCGACAGCCTAAATGATCCCC-3' sFc.gamma.RIII
5'-AAAAAAACATATGCGGACTGAAG-3' 5'-AAAAAAGCTTAACCTTGAGTGATG-3'
sFc.epsilon.RIa 5'-GATGGCCATATGGCAGTCCCTCAG-3'
5'-CAATGGATCCTAAAATTGTAGCCAG-3' sFc.epsilon.RII
5'-AAAAAAACATATGGAGTTGCAGG-3' 5'-TGGCTGGATCCATGCTCAAG-3'
[0139] Introduced restriction sites are underlined, start- and
stop-codons are depicted as bold-italics
TABLE-US-00006 TABLE 6 Refolding Conditions for the FcRs Construct
Buffer sFc.gamma.RI 0.1 M TRIS/HCI, 1.2M arginine, 150 mM NaCI, S
mM GSH, 0.5 mM GSSG, 0.02% sodium azide, pH 8.0 sFc.gamma.RIIa 0.1
M TRIS/HCI, 1.4M arginine, 150 mM NaCI, 2 mM GSH, 0.5 mM GSSG,
0.02% sodium azide, pH 8.0 sFc.gamma.RIIb 0.1 M TRIS/HCI, 1.4M
arginine, 150 mM NaCI, S mM GSH, 0.5 mM GSSG, 0.02% sodium azide,
pH 8.0 sFc.gamma.RIII 0.1 M TRIS/HCI, 1.0M arginine, 150 mM NaCI, 2
mM GSH, 0.5 mM GSSG, 0.02% sodium azide, pH 8.0 sFc.epsilon.RII 0.1
M TRIS/HCl, a.8M arginine, 150 mM NaCI, 5 mM GSH, 0.5 mM GSSG,
0.02% sodium azide, pH 8.3
TABLE-US-00007 TABLE 7 Crystallisation Conditions for the FcRs
Space group, cell Resolu- Construct Condition constants tion
sFc.gamma.RIIa 26% PEG 8000, C2, a = 80.4 .ANG., 3.0 .ANG. 0.2M
sodium b = 49.7 .ANG., c = 54.6 .ANG., acetate/acetic a = g =
90.degree., b = 128.1.degree. acid pH 4.6, 0.02% sodium azide
sFc.gamma.RIIb 33% PEG 2000, P212121, a = 40.8 .ANG., 1.7 .ANG.
0.2M sodium b = 50.9 .ANG., c = 80.5 .ANG., acetate, 0.02% a = b =
g = 90.degree. sodium azide, pH 5.4 sFc.gamma.RIII 22% PEG 8000,
P22121, a = 36.7 .ANG., 2.5 .ANG. 0.1M MES/TRIS pH b = 60.3 .ANG.,
c = 85.6 .ANG., 7.8, 0.02% sodium a = b = g = 90.degree. azide
sFc.gamma.RIII: 6% PEG 8000, 0.1M P6522, 3.3 .ANG. hFc1 MES/TRIS pH
5.6, a = b = 115.0 .ANG., 0.2M Na/K c = 303.3 .ANG., tartrate,
0.02% a = b = 90.degree., g = 120.degree. sodium azide
sFc.gamma.RIII 22% PEG 8000, P22121, a = 36.7 .ANG., 2.5 .ANG. 0.1M
MES/TRIS pH b = 60.3 .ANG., c = 85.6 .ANG., 7.8, 0.02% sodium a = b
= g = 90.degree. azide
REFERENCES
[0140] Ades, E. W., Phillips, D. J., Shore, S. L., Gordon, D. S.,
LaVia, M. F., Black, C. M., Reimer, C. B. (1976), Analysis of
mononuclear cell surfaces with fluoresceinated Staphylococcal
protein A complexed with IgG antibody or heat-aggregated
.gamma.-globulin, J. Immunol. 117, 2119. [0141] Allen J. M., Seed
B.; "Nucleotide sequence of three cDNAs for the human high affinity
Fc receptor (FcRI)"; Nucleic Acids Res. 16: 11824-11824 (1988).
[0142] Amigorena, S., Bonnerot, C., Drake, J. R., Choquet, D.,
Hunziker, W., Guillet, J. G., Webster, P., Sautes, C., Mellman, I.,
Fridman, W. H. (1992), Cytoplasmic domain heterogeneity and
functions of IgG Fc receptors in B lymphocytes, Science 256,
1808-1812. [0143] Barton, G. C. (1993), ALSCRIPT: tool to format
multiple sequence alignments, Prot. Eng. 6, 37-40. [0144] Bazil, V.
and Strominger, J. L. (1994), Metalloprotease and serine protease
are involved in cleavage of CD43, CD44, and CD16 from stimulated
human granulocytes, J. Immunol. 152, 1314-1322. [0145] Brunger, A.
T., Kuriyan, J., Karplus, M. (1987), Crystallographic R factor
refinement by molecular dynamics, Science 35, 458-460. [0146]
Burmeister, W. P., Huber, A. H., Bjorkman, P. J. (1994), Crystal
structure of the complex of rat neonatal Fc receptor with Fc,
Nature 372, 379-383. [0147] Ceuppens, J. L., Baroja, M. L., van
Vaeck, F., Anderson, C. L. (1988), Defect in the membrane
expression of high affinity 72 kD Fc.gamma. receptors on phagocytic
cells in four healthy subjects, J. Clin. Invest. 82, 571-578.
[0148] Collaborative computational project, Number 4 (1994), The
CCP4 suite: Programs for protein crystallography, Acta crystallogr.
D50, 760-763. [0149] Deisenhofer, J., Jones, T. A., Huber, R.,
Sjodahl, J., Sjoquist, J. (1978), Crystallization, crystal
structure analysis and atomic model of the complex formed by a
human Fc fragment and fragment B of protein A from Staphylococcus
aureus, Z. Phys. Chem. 359, 975-985. [0150] Deisenhofer, J. (1981),
Crystallographic refinement and atomic models of a human Fc
fragment and its complex with fragment B of protein A from
Staphylococcus aureus at 2.9- and 2.8 A resolution, Biochemistry
20, 2361-2370. [0151] Deisenhofer J., Colman P M., Huber R., Haupt
H., Schwick G.; "Crystallographic structural studies of a human
Fc-fragment. I. An electrondensity map at 4 .ANG. resolution and a
partial model"; Hoppe-Seyler's Z. Physiol. Chem. 357:435-445
(1976). [0152] Dulau, L., Cheyrou, A., Aigle, M. (1989), Directed
mutagenesis using PCR, Nucleic Acids Res. 17, 2873. [0153] Ellman
(1959), Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82,
79-77. [0154] Engelhardt, W., Geerds, C., Frey, J. (1990),
Distribution, inducibility and biological function of the cloned
and expressed human .beta.Fc receptor II, Eur. J. Immunol. 20,
1367-1377. [0155] Engh, R. A. and Huber, R. (1991), Accurate bond
and angle parameters for X-ray protein structure refinement, Acta
crystallogr. A47, 392-400. [0156] Fleit, H. B., Kobasiuk, C. D.,
Daly, C., Furie, R., Levy, P. C., Webster, R. O. (1992), A soluble
form of Fc.gamma.RIII is present in human serum and other body
fluids and is elevated at sites of inflammation, Blood 79,
2721-2728. [0157] Fridman, W. H., Bonnerot, C., Daeron, M.,
Amigorena, S., Teillaud, J.-L., Sautes, C. (1992), Structural bases
of Fc.gamma. receptor functions, Immunol. Rev. 125, 49-76. [0158]
Fridman, W. H., Teillaud, J.-L., Bouchard, C., Teillaud, C.,
Astier, A., Tartour, E., Galon, J., Mathiot, C., Sautes, C. (1993),
Soluble Fc.gamma. receptors, J. Leukocyte Biol. 54, 504-512. [0159]
Gabb, H. A., Jackson, R. M., Sternberg, M. J. E. (1997), Modelling
protein docking using shape complementarity, electrostatics and
biochemical information, J. Mol. Biol. 272, 106-120. [0160] Galon,
J., Bouchard, C., Fridman, W. H., Sautes, C. (1995), Ligands and
biological activities of soluble Fc.gamma. receptors, Immunol.
Lett. 44, 175-181. [0161] Genetics Compouter Group (1994), Program
Manual for the Wisconsin Package Version 8, Madison, Wis. [0162]
Gordon, J. et al., (1980), The molecules controlling B lymphocytes.
Immunol. Today, 8: 339-344. [0163] Grodberg, J. and Dunn, J. J.
(1988), OmpT encodes the Escherichia coli outer membrane protease
that cleaves T7 RNA polymerase during purification, J. Bacteriol.
170, 1245-1253. [0164] Hogarth, P. M., Hulett, M. D., lerino, F.
L., Tate, B., Powell, M. S., Brinkworth, R. I. (1992),
Identification of the immunoglobulin binding regions (IBR) of
Fc.gamma.RII and FC.epsilon.RI, Immunol. Rev. 125, 21-35. [0165]
Homsy, J., Meyer, M., Tateno, M., Clarkson, S.; Levy, J. A. (1989),
The Fc and not CD4 receptor mediates antibody enhancement of HIV
infection in human cells, Science 244, 1357-1360 [0166] Hoover, R.
G., Lary, C., Page, R., Travis, P., Owens, R., Flick, J.,
Kornbluth, J., Barlogie, B. (1995), Autoregulatory circuits in
myeloma: Tumor cell cytotoxity mediated by soluble CD16, J. Clin.
Invest. 95, 241-247. [0167] Huber, R., Deisenhofer, J., Colman, P.
M., Matsushima, M. and Palm, W. (1976), Crystallographic structure
studies of an IgG molecule and an Fc fragment, Nature 264, 415-420.
[0168] Hulett, M. D., Witort, E., Brinkworth, R. I., McKenzie, I.
F. C., Hogarth, P. M. (1994), Identification of the IgG binding
site of the human low affinity receptor for IgG Fc.gamma.RII, J.
Biol. Chem. 269, 15287-15293. [0169] Hulett, M. D., Witort, E.,
Brinkworth, R. I., McKenzie, I. F. e., Hogarth, P. M. (1995),
Multiple regions of human Fc.gamma.RII (C032) contribute to the
binding of IgG, J. Biol. Chem. 270, 21188-21194. [0170] Ierino, F.
L., Powell, M. S., McKenzie, I. F. C., Hogarth, P. M. (1993),
Recombinant soluble human Fc.gamma.RII: Production,
characterization, and inhibition of the arthus reaction, J. Exp.
Med. 178, 1617-1628. [0171] Jancarik, J. and Kim, S. H. (1991),
Sparse matrix sampling: A screening method for crystallization of
proteins, J. Appl. Crystallogr. 24, 409-411. [0172] Jones, T. A.,
Zou, J.-Y., Cowan, S. W., Kjeldgaard, M. (1991), Improved methods
for building protein models in electron density maps and the
location of errors in these models, Acta crystallogr. A47, 110-119.
[0173] Kikutani H., Inui S., Sato R., Sarsumian E. L., Owaki H.,
Yamasaki K., Kaisho T., Uchibayashi N., Hardy R. R., Hirano T.,
Tsunasawa S., Sakiyama F., Suemura M., Kishimoto T.; "Molecular
structure of human lymphocyte receptor for immunoglobulin E"; Cell
47(5):657-665 (1986). [0174] Khayat, D., Soubrane, C., Andriew, J.
M., Visonneau, S., Eme, D., Tourani, J. M., Beldjord, K., Weil, M.,
Fernandez, E., Jaquillat, C. (1990), Changes of soluble CD16 levels
in serum of HIV patients: Correlation with clinical and biological
prognostic factors, J. Infect. Dis. 161, 430-435. [0175] Kochan J.,
Pettine L. F., Hakimi J., Kishi K., Kinet J. P.; "Isolation of the
gene coding for the alpha subunit of the human high affinity IgE
receptor"; Nucleic Acids Res. 16:3584-3584 (1988). [0176] Simmons
D., Seed B.; "The Fc-gamma receptor of natural killer cells is a
phospholipid-linked membrane protein"; Nature 333:568-570 (1988).
[0177] Kraulis, P. J. (1991), MOLSCRIPT: a program to produce both
detailed and schematic plots of protein structures, J. Appl. Cryst.
24, 946-950. [0178] Leslie, A. G. W. (1997), Mosflm user guide,
mosflm version 5.50, MRC Laboratory of Molecular Biology,
Cambridge, UK. [0179] Lessel, U. and Schomburg, D. (1994),
Similarities between protein 3-D structures, Protein Eng. 7,
1175-1187. [0180] Littaua, R., Kurane, I. and Ennis, F. A. (1990),
Human IgG Fc receptor II mediates antibody-dependent enhancement of
dengue virus infection, J. Immunol. 144, 3183-3186. [0181] Lynch,
R. G., Hagen, M., Mueller, A., Sandor, M. (1995), Potential role of
Fc.gamma.R in early development of murine lymphoid cells: Evidence
for functional interaction between Fc.gamma.R on pre-thymocytes and
an alternative, non-Ig ligand on thymic stromal cells, Immunol.
Lett. 44, 105-109. [0182] Mathiot, C., Teillaud, J. L., Elmalek,
M., Mosseri, L., Euller-Ziegler, L., Daragon, A., Grosbois, B.,
Michaux, J. L., Facon, T., Bernard, J. F., Duclos, B., Monconduit.
M., Fridman, W. H. (1993), Correlation between serum soluble CD16
(sCD16) levels and disease stage in patients with multiple myeloma,
J. Clin. Immunol. 13, 41-48. [0183] Merritt, E. A. and Murphy. M.
E. P. (1994), Raster3D Version 2.0. A program for photorealistic
molecular graphics, Acta Cryst. D50, 869-873. [0184] Metzger, H.
(1992A), Transmembrane signaling: The joy of aggregation, J.
Immunol. 149, 1477-1487. [0185] Metzger, H. (1992B), The receptor
with high affinity for Ig E, Immunol. Rev. 125, 37-48. [0186]
Muller, S, and Hoover, R. G. (1985), T cells with Fc receptors in
myeloma; suppression of growth and secretion of MOPC-315 by T alpha
cells, J. Immunol. 134, 644-7. [0187] Nicholls, A., Sharp, K. A.,
Honig, B. (1991), Protein folding and association: insights from
the interfacial and thermodynamic properties of hydrocarbons,
Proteins 11, 281-296. [0188] Poo, H., Kraus, J. C., Mayo-Bond, L.,
Todd, R. F., Petty, H. R. (1995), Interaction of Fc.gamma. receptor
IIIB with complement receptor type 3 in fibroblast transfectants:
evidence from lateral diffusion and resonance energy transfer
studies, J. Mol. Biol. 247, 597-603. [0189] Rappaport, E. F.,
Cassel, D. L., Walterhouse, D. O., McKenzie, S. E., Surrey, S.,
Keller, M. A., Schreiber, A. D., Schwartz, E. (1993), A soluble
form of the human Fc receptor Fc.gamma.RIIa: cloning, transcript
analysis and detection. Exp. Hematol. 21, 689-696. [0190] Ravanel,
K., Castelle, C., Defrance, T., Wild, T. F., Charron, D., Lotteau,
V., Rabourdincombe, C. (1997), Measles virus nucleocapsid protein
binds to Fc.gamma.RII and inhibits human B cell antibody
production. J. Exp. Med. 186, 269-278. [0191] Roman, S., Moore, J.
S., Darby, C., Muller, S., Hoover, R. G. (1988), Modulation of Ig
gene expression by Ig binding factors. Suppression of alpha-H chain
and lambda-2-L chain mRNA accumulation in MOPC-315 by IgA-binding
factor, J. Immunology 140, 3622-30. [0192] Sarfat, D. et al.,
(1988), Elevation of IgE-binding factors of serum in patients with
B-cell derived chronic lymphocytic leukemia. Blood, 71: 94-98.
[0193] Sauer-Eriksson, A. E., Kleywegt, G. J., Uhlen, M., Jones, T.
A. (1995), Crystal structure of the C2 fragment of streptococcal
protein G in complex with the Fc domain of human IgG, Structure 3,
265-78. [0194] Small, T., et al., (1990), B-cell differentiation
following autologous, conventional or T-cell depleted bone marrow
transplantation: a recapitulation of normal B-cell ontogeny. Blood,
76: 1647-1656. [0195] Sondermann, P., Huber, R., Jacob, U. (1998B),
Preparation and crystallization of active soluble human
Fc.gamma.RIIb derived from E. coli, Protein Structure, submitted.
[0196] Sondermann, P., Kutscher, C., Jacob, U., Frey, J. (1998A),
Characterization and crystallization of soluble human Fc.gamma.
receptor 11 isoforms produced in insect cells, Biochemistry,
submitted. [0197] Sondermann, P., Kutscher, C., Jacob, U., Frey,
J., Analysis of complexes of IgG and soluble human
Fc.gamma.-Receptor II produced in insect cells and its
crystallization, submitted. [0198] Stengelin S., Stamenkovic I.,
Seed B.; "Isolation of cDNAs for two distinct human Fc receptors by
ligand affinity cloning"; EMBO J. 7:1053-1059 (1988). [0199] Tax,
W. J. M., Willems, H. W., Reekers, P. P. M., Capel, P. J. A.,
Koene, R. A. P. (1983), Polymorphism in mitogenic effect of IgG1
monoclonal antibodies against T3 antigen on human T cells, Nature
304, 445-447. [0200] Teillaud, J. L., Brunati, S., Elmalek, M.,
Astier, A., Nicaise, P., Moncuit, J., Mathiot, C., Job-Deslandre,
C., Fridman, W. H. (1990), Involvement of FcR+T cells and of IgG-BF
in the control of myeloma cells, Mol. Immunol. 27, 1209-17. [0201]
Turk, D. (1992), Ph.D. Thesis, T U Munchen, Germany. [0202]
Ulvestad, E., Matre, R., Tonder, O. (1988), IgG Fc receptors in
sera from patients with Rheumatoid Arthritis and Systemic Lupus
Erythematosus, Scand. J. Rheumatol., Suppl. 75, 203-208. [0203] van
de Winkel, J. G. J. and Capel, P. J. A. (1993), Human IgG Fc
receptor heterogeneity: Molecular aspects and clinical
implications, Immunol. Today 14, 215-221. [0204] Varin, N., Sautes,
C., Galinha, A., Even, J., Hogarth, P. M., Fridman, W. H. (1989),
Recombinant soluble receptors for the Fc.gamma. portion inhibit
antibody production in vitro, Eur. J. Immunol. 19, 2263-2268.
[0205] Yang, Z., Delgado, R., Xu, L., Todd, R. F., Nabel, E. G.,
Sanchez, A. Nabel, G. J. (1998), Distinct cellular interactions of
secreted and transmembrane Ebola virus glycoproteins, Science 279,
983-984. [0206] Zhou, M.-J., Todd, R. F., van de Winkel, J. G. J.,
Petty, H. R. (1993), Cocapping of the leukoadhesin molecules
complement receptor type 3 and lymphocyte function-associated
antigen-1 with Fc.gamma. receptor III on human neutrophils.
Possible role of lectin-like interactions, J. Immunol. 150,
3030-3041.
Sequence CWU 1
1
201269PRTHomo sapiens 1Met Ala Val Ile Ser Leu Gln Pro Pro Trp Val
Ser Val Phe Gln Glu1 5 10 15Glu Thr Val Thr Leu His Cys Glu Val Leu
His Leu Pro Gly Ser Ser 20 25 30Ser Thr Gln Trp Phe Leu Asn Gly Thr
Ala Thr Gln Thr Ser Thr Pro 35 40 45Ser Tyr Arg Ile Thr Ser Ala Ser
Val Asn Asp Ser Gly Glu Tyr Arg 50 55 60Cys Gln Arg Gly Leu Ser Gly
Arg Ser Asp Pro Ile Gln Leu Glu Ile65 70 75 80His Arg Gly Trp Leu
Leu Leu Gln Val Ser Ser Arg Val Phe Thr Glu 85 90 95Gly Glu Pro Leu
Ala Leu Arg Cys His Ala Trp Lys Asp Lys Leu Val 100 105 110Tyr Asn
Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe Lys Phe Phe His 115 120
125Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile Ser His Asn Gly
130 135 140Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr Thr Ser
Ala Gly145 150 155 160Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala
Pro Val Leu Asn Ala 165 170 175Ser Val Thr Ser Pro Leu Leu Glu Gly
Asn Leu Val Thr Leu Ser Cys 180 185 190Glu Thr Lys Leu Leu Leu Gln
Arg Pro Gly Leu Gln Leu Tyr Phe Ser 195 200 205Phe Tyr Met Gly Ser
Lys Thr Leu Arg Gly Arg Asn Thr Ser Ser Glu 210 215 220Tyr Gln Ile
Leu Thr Ala Arg Arg Glu Asp Ser Gly Leu Tyr Trp Cys225 230 235
240Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg Ser Pro Glu Leu
245 250 255Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val 260
2652174PRTHomo sapiens 2Met Ala Ala Pro Pro Lys Ala Val Leu Lys Leu
Glu Pro Pro Trp Ile1 5 10 15Asn Val Leu Gln Glu Asp Ser Val Thr Leu
Thr Cys Gln Gly Ala Arg 20 25 30Ser Pro Glu Ser Asp Ser Ile Gln Trp
Phe His Asn Gly Asn Leu Ile 35 40 45Pro Thr His Thr Gln Pro Ser Tyr
Arg Phe Lys Ala Asn Asn Asn Asp 50 55 60Ser Gly Glu Tyr Thr Cys Gln
Thr Gly Gln Thr Ser Leu Ser Asp Pro65 70 75 80Val His Leu Thr Val
Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His 85 90 95Leu Glu Phe Gln
Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser Trp 100 105 110Lys Asp
Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys Ser 115 120
125Gln Lys Phe Ser Arg Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala Asn
130 135 140His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile Gly
Tyr Thr145 150 155 160Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val
Gln Val Pro 165 1703185PRTHomo sapiens 3Met Gly Thr Pro Ala Ala Pro
Pro Lys Ala Val Leu Lys Leu Glu Pro1 5 10 15Gln Trp Ile Asn Val Leu
Gln Glu Asp Ser Val Thr Leu Thr Cys Arg 20 25 30Gly Thr His Ser Pro
Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly 35 40 45Asn Leu Ile Pro
Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn 50 55 60Asn Asn Asp
Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu65 70 75 80Ser
Asp Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln 85 90
95Thr Pro His Leu Glu Phe Gln Glu Gly Glu Thr Ile Val Leu Arg Cys
100 105 110His Ser Trp Lys Asp Lys Pro Leu Val Lys Val Thr Phe Phe
Gln Asn 115 120 125Gly Lys Ser Lys Lys Phe Ser Arg Ser Asp Pro Asn
Phe Ser Ile Pro 130 135 140Gln Ala Asn His Ser His Ser Gly Asp Tyr
His Cys Thr Gly Asn Ile145 150 155 160Gly Tyr Thr Leu Tyr Ser Ser
Lys Pro Val Thr Ile Thr Val Gln Ala 165 170 175Pro Ser Ser Ser Pro
Met Gly Ile Ile 180 1854176PRTHomo sapiens 4Met Arg Thr Glu Asp Leu
Pro Lys Ala Val Val Phe Leu Glu Pro Gln1 5 10 15Trp Tyr Ser Val Leu
Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly 20 25 30Ala Tyr Ser Pro
Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser 35 40 45Leu Ile Ser
Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val 50 55 60Asn Asp
Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser65 70 75
80Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala
85 90 95Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys
His 100 105 110Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu
Gln Asn Gly 115 120 125Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp
Phe His Ile Pro Lys 130 135 140Ala Thr Leu Lys Asp Ser Gly Ser Tyr
Phe Cys Arg Gly Leu Val Gly145 150 155 160Ser Lys Asn Val Ser Ser
Glu Thr Val Asn Ile Thr Ile Thr Gln Gly 165 170 1755183PRTHomo
sapiens 5Met Ala Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro
Trp Asn1 5 10 15Arg Ile Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn
Gly Asn Asn 20 25 30Phe Phe Glu Val Ser Ser Thr Lys Trp Phe His Asn
Gly Ser Leu Ser 35 40 45Glu Glu Thr Asn Ser Ser Leu Asn Ile Val Asn
Ala Lys Phe Glu Asp 50 55 60Ser Gly Glu Tyr Lys Cys Gln His Gln Gln
Val Asn Glu Ser Glu Pro65 70 75 80Val Tyr Leu Glu Val Phe Ser Asp
Trp Leu Leu Leu Gln Ala Ser Ala 85 90 95Glu Val Val Met Glu Gly Gln
Pro Leu Phe Leu Arg Cys His Gly Trp 100 105 110Arg Asn Trp Asp Val
Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala 115 120 125Leu Lys Tyr
Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr 130 135 140Val
Glu Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu145 150
155 160Asp Tyr Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro
Arg 165 170 175Glu Lys Tyr Trp Leu Gln Phe 1806275PRTHomo sapiens
6Met Asp Thr Thr Gln Ser Leu Lys Gln Leu Glu Glu Arg Ala Ala Arg1 5
10 15Asn Val Ser Gln Val Ser Lys Asn Leu Glu Ser His His Gly Asp
Gln 20 25 30Met Thr Gln Lys Ser Gln Ser Thr Gln Ile Ser Gln Glu Leu
Glu Glu 35 40 45Leu Arg Ala Glu Gln Gln Arg Leu Lys Ser Gln Asp Leu
Glu Leu Ser 50 55 60Trp Asn Leu Asn Gly Leu Gln Ala Asp Leu Ser Ser
Phe Lys Ser Gln65 70 75 80Glu Leu Asn Glu Arg Asn Glu Ala Ser Asp
Leu Leu Glu Arg Leu Arg 85 90 95Glu Glu Val Thr Lys Leu Arg Met Glu
Leu Gln Val Ser Ser Gly Phe 100 105 110Val Cys Asn Thr Cys Pro Glu
Lys Trp Ile Asn Phe Gln Arg Lys Cys 115 120 125Tyr Tyr Phe Gly Lys
Gly Thr Lys Gln Trp Val His Ala Arg Tyr Ala 130 135 140Cys Asp Asp
Met Glu Gly Gln Leu Val Ser Ile His Ser Pro Glu Glu145 150 155
160Gln Asp Phe Leu Thr Lys His Ala Ser His Thr Gly Ser Trp Ile Gly
165 170 175Leu Arg Asn Leu Asp Leu Lys Gly Glu Phe Ile Trp Val Asp
Gly Ser 180 185 190His Val Asp Tyr Ser Asn Trp Ala Pro Gly Glu Pro
Thr Ser Arg Ser 195 200 205Gln Gly Glu Asp Cys Val Met Met Arg Gly
Ser Gly Arg Trp Asn Asp 210 215 220Ala Phe Cys Asp Arg Lys Leu Gly
Ala Trp Val Cys Asp Arg Leu Ala225 230 235 240Thr Cys Thr Pro Pro
Ala Ser Glu Gly Ser Ala Glu Ser Met Gly Pro 245 250 255Asp Ser Arg
Pro Asp Pro Asp Gly Arg Leu Pro Thr Pro Ser Ala Pro 260 265 270Leu
His Ser 2757820DNAHomo sapiens 7catatggcag tgatctcttt gcagcctcca
tgggtcagcg tgttccaaga ggaaaccgta 60accttgcact gtgaggtgct ccatctgcct
gggagcagct ctacacagtg gtttctcaat 120ggcacagcca ctcagacctc
gacccccagc tacagaatca cctctgccag tgtcaatgac 180agtggtgaat
acaggtgcca gagaggtctc tcagggcgaa gtgaccccat acagctggaa
240atccacagag gctggctact actgcaggtc tccagcagag tcttcacgga
aggagaacct 300ctggccttga ggtgtcatgc gtggaaggat aagctggtgt
acaatgtgct ttactatcga 360aatggcaaag cctttaagtt tttccactgg
aattctaacc tcaccattct gaaaaccaac 420ataagtcaca atggcaccta
ccattgctca ggcatgggaa agcatcgcta cacatcagca 480ggaatatctg
tcactgtgaa agagctattt ccagctccag tgctgaatgc atctgtgaca
540tccccactcc tggaggggaa tctggtcacc ctgagctgtg aaacaaagtt
gctcttgcag 600aggcctggtt tgcagcttta cttctccttc tacatgggca
gcaagaccct gcgaggcagg 660aacacatcct ctgaatacca aatactaact
gctagaagag aagactctgg gttatactgg 720tgcgaggctg ccacagagga
tggaaatgtc cttaagcgca gccctgagtt ggagcttcaa 780gtgcttggcc
tccagttacc aactcctgtc tagtctcgag 8208533DNAHomo sapiens 8catatggcag
ctcccccaaa ggctgtgctg aaacttgagc ccccgtggat caacgtgctc 60caggaggact
ctgtgactct gacatgccag ggggctcgca gccctgagag cgactccatt
120cagtggttcc acaatgggaa tctcattccc acccacacgc agcccagcta
caggttcaag 180gccaacaaca atgacagcgg ggagtacacg tgccagactg
gccagaccag cctcagcgac 240cctgtgcatc tgactgtgct ttccgaatgg
ctggtgctcc agacccctca cctggagttc 300caggagggag aaaccatcat
gctgaggtgc cacagctgga aggacaagcc tctggtcaag 360gtcacattct
tccagaatgg aaaatcccag aaattctccc gtttggatcc caccttctcc
420atcccacaag caaaccacag tcacagtggt gattaccact gcacaggaaa
cataggctac 480acgctgttct catccaagcc tgtgaccatc actgtccaag
tgccctgaag ctt 5339569DNAHomo sapiens 9ccatggggac acctgcagct
cccccaaagg ctgtgctgaa actcgagccc cagtggatca 60acgtgctcca ggaggactct
gtgactctga catgccgggg gactcacagc cctgagagcg 120actccattca
gtggttccac aatgggaatc tcattcccac ccacacgcag cccagctaca
180ggttcaaggc caacaacaat gacagcgggg agtacacgtg ccagactggc
cagaccagcc 240tcagcgaccc tgtgcatctg actgtgcttt ctgagtggct
ggtgctccag acccctcacc 300tggagttcca ggagggagaa accatcgtgc
tgaggtgcca cagctggaag gacaagcctc 360tggtcaaggt cacattcttc
cagaatggaa aatccaagaa attttcccgt tcggatccca 420acttctccat
cccacaagca aaccacagtc acagtggtga ttaccactgc acaggaaaca
480taggctacac gctgtactca tccaagcctg tgaccatcac tgtccaagct
cccagctctt 540caccgatggg gatcatttag gctgtcgac 56910538DNAHomo
sapiens 10catatgcgga ctgaagatct cccaaaggct gtggtgttcc tggagcctca
atggtacagc 60gtgcttgaga aggacagtgt gactctgaag tgccagggag cctactcccc
tgaggacaat 120tccacacagt ggtttcacaa tgagagcctc atctcaagcc
aggcctcgag ctacttcatt 180gacgctgcca cagtcaacga cagtggagag
tacaggtgcc agacaaacct ctccaccctc 240agtgacccgg tgcagctaga
agtccatatc ggctggctgt tgctccaggc ccctcggtgg 300gtgttcaagg
aggaagaccc tattcacctg aggtgtcaca gctggaagaa cactgctctg
360cataaggtca catatttaca gaatggcaaa gacaggaagt attttcatca
taattctgac 420ttccacattc caaaagccac actcaaagat agcggctcct
acttctgcag ggggcttgtt 480gggagtaaaa atgtgtcttc agagactgtg
aacatcacca tcactcaagg ttaagctt 53811560DNAHomo sapiens 11catatggcag
tccctcagaa acctaaggtc tccttgaacc ctccatggaa tagaatattt 60aaaggagaga
atgtgactct tacatgtaat gggaacaatt tctttgaagt cagttccacc
120aaatggttcc acaatggcag cctttcagaa gagacaaatt caagtttgaa
tattgtgaat 180gccaaatttg aagacagtgg agaatacaaa tgtcagcacc
aacaagttaa tgagagtgaa 240cctgtgtacc tggaagtctt cagtgactgg
ctgctccttc aggcctctgc tgaggtggtg 300atggagggcc agcccctctt
cctcaggtgc catggttgga ggaactggga tgtgtacaag 360gtgatctatt
ataaggatgg tgaagctctc aagtactggt atgagaacca caacatctcc
420attacaaatg ccacagttga agacagtgga acctactact gtacgggcaa
agtgtggcag 480ctggactatg agtctgagcc cctcaacatt actgtaataa
aagctccgcg tgagaagtac 540tggctacaat tttaggatcc 56012532DNAHomo
sapiens 12catatggagt tgcaggtgtc cagcggcttt gtgtgcaaca cgtgccctga
aaagtggatc 60aatttccaac ggaagtgcta ctacttcggc aagggcacca agcagtgggt
ccacgcccgg 120tatgcctgtg acgacatgga agggcagctg gtcagcatcc
acagcccgga ggagcaggac 180ttcctgacca agcatgccag ccacaccggc
tcctggattg gccttcggaa cttggacctg 240aagggggagt ttatctgggt
ggatgggagc cacgtggact acagcaactg ggctccaggg 300gagcccacca
gccggagcca gggcgaggac tgcgtgatga tgcggggctc cggtcgctgg
360aacgacgcct tctgcgaccg taagctgggc gcctgggtgt gcgaccggct
ggccacatgc 420acgccgccag ccagcgaagg ttccgcggag tccatgggac
ctgattcaag accagaccct 480gacggccgcc tgcccacccc ctctgcccct
ctccactctt gagcatggat cc 532131419DNAHomo sapiens 13ggctgtgact
gctgtgctct gggcgccact cgctccaggg agtgatggga atcctgtcat 60ttttacctgt
ccttgccact gagagtgact gggctgactg caagtccccc cagccttggg
120gtcatatgct tctgtggaca gctgtgctat tcctggctcc tgttgctggg
acacctgcag 180ctcccccaaa ggctgtgctg aaactcgagc cccagtggat
caacgtgctc caggaggact 240ctgtgactct gacatgccgg gggactcaca
gccctgagag cgactccatt cagtggttcc 300acaatgggaa tctcattccc
acccacacgc agcccagcta caggttcaag gccaacaaca 360atgacagcgg
ggagtacacg tgccagactg gccagaccag cctcagcgac cctgtgcatc
420tgacagtgct ttctgagtgg ctggtgctcc agacccctca cctggagttc
caggagggag 480aaaccatcgt gctgaggtgc cacagctgga aggacaagcc
tctggtcaag gtcacattct 540tccagaatgg aaaatccaag aaattttccc
gttcggatcc caacttctcc atcccacaag 600caaaccacag tcacagtggt
gattaccatt gcacaggaaa cataggctac acgctgtact 660catccaagcc
tgtgaccatc actgtccaag ctcccagctc ttcaccgatg gggatcattg
720tggctgtggt cactgggatt gctgtagctg ccattgttgc tgctgtagtg
gccttgatct 780actgcaggaa aaagcggatt tcagccaatc ccactaatcc
tgatgaggct gacaaagttg 840gggctgagaa cacaatcacc tattcacttc
tcatgcaccc ggatgctctg gaagagcctg 900atgaccagaa ccgtatttag
tctccattgt cttgcattgg gatttgagaa gaaatcagag 960agggaagatc
tggtatttcc tggcctaaat tccccttggg gaggacaggg agatgctgca
1020gttccaaaag agaaggtttc ttccagagtc atctacctga gtcctgaagc
tccctgtcct 1080gaaagccaca gacaatatgg tcccaaatgc ccgactgcac
cttctgtgct tcagctcttc 1140ttgacatcaa ggctcttccg ttccacatcc
acacagccaa tccaattaat caaaccactg 1200ttattaacag ataatagcaa
cttgggaaat gcttatgtta caggttacgt gagaacaatc 1260atgtaaatct
atatgatttc agaaatgtta aaatagacta acctctacca gcacattaaa
1320agtgattgtt tctgggtgat aaaattattg atgattttta ttttctttat
ttttctataa 1380agatcatata ttacttttat aataaaacat tataaaaac
1419141068DNAHomo sapiens 14agatctcagc acagtaagca ccaggagtcc
atgaagaaga tggctcctgc catggaatcc 60cctactctac tgtgtgtagc cttactgttc
ttcgctccag atggcgtgtt agcagtccct 120cagaaaccta aggtctcctt
gaaccctcca tggaatagaa tatttaaagg agagaatgtg 180actcttacat
gtaatgggaa caatttcttt gaagtcagtt ccaccaaatg gttccacaat
240ggcagccttt cagaagagac aaattcaagt ttgaatattg tgaatgccaa
atttgaagac 300agtggagaat acaaatgtca gcaccaacaa gttaatgaga
gtgaacctgt gtacctggaa 360gtcttcagtg actggctgct ccttcaggcc
tctgctgagg tggtgatgga gggccagccc 420ctcttcctca ggtgccatgg
ttggaggaac tgggatgtgt acaaggtgat ctattataag 480gatggtgaag
ctctcaagta ctggtatgag aaccacaaca tctccattac aaatgccaca
540gttgaagaca gtggaaccta ctactgtacg ggcaaagtgt ggcagctgga
ctatgagtct 600gagcccctca acattactgt aataaaagct ccgcgtgaga
agtactggct acaatttttt 660atcccattgt tggtggtgat tctgtttgct
gtggacacag gattatttat ctcaactcag 720cagcaggtca catttctctt
gaagattaag agaaccagga aaggcttcag acttctgaac 780ccacatccta
agccaaaccc caaaaacaac tgatataatt aactcaagaa atatttgcaa
840cattagtttt tttccagcat cagcaattgc tactcaattg tcaaacacag
cttgcaatat 900acatagaaac gtctgtgctc aaggatttat agaaatgctt
cattaaactg agtgaaactg 960attaagtggc atgtaatagt aagtgctcaa
ttaacattgg ttgaataaat gagagaatga 1020atagattcat ttattagcat
ttgtaaaaga gatgttcaat ttagatct 1068151321DNAHomo sapiens
15gacagatttc actgctccca ccagcttgga gacaacatgt ggttcttgac aactctgctc
60ctttgggttc cagttgatgg gcaagtggac accacaaagg cagtgatctc tttgcagcct
120ccatgggtca gcgtgttcca agaggaaacc gtaaccttgc actgtgaggt
gctccatctg 180cctgggagca gctctacaca gtggtttctc aatggcacag
ccactcagac ctcgaccccc 240agctacagaa tcacctctgc cagtgtcaat
gacagtggtg aatacaggtg ccagagaggt 300ctctcagggc gaagtgaccc
catacagctg gaaatccaca gaggctggct actactgcag 360gtctccagca
gagtcttcac ggaaggagaa cctctggcct tgaggtgtca tgcgtggaag
420gataagctgg tgtacaatgt gctttactat cgaaatggca aagcctttaa
gtttttccac 480tggaattcta acctcaccat tctgaaaacc aacataagtc
acaatggcac ctaccattgc 540tcaggcatgg gaaagcatcg ctacacatca
gcaggaatat ctgtcactgt gaaagagcta 600tttccagctc cagtgctgaa
tgcatctgtg acatccccac tcctggaggg gaatctggtc 660accctgagct
gtgaaacaaa gttgctcttg cagaggcctg gtttgcagct ttacttctcc
720ttctacatgg gcagcaagac cctgcgaggc aggaacacat cctctgaata
ccaaatacta 780actgctagaa gagaagactc tgggttatac tggtgcgagg
ctgccacaga ggatggaaat 840gtccttaagc gcagccctga gttggagctt
caagtgcttg gcctccagtt accaactcct 900gtctggtttc atgtcctttt
ctatctggca gtgggaataa tgtttttagt gaacactgtt 960ctctgggtga
caatacgtaa
agaactgaaa agaaagaaaa agtgggattt agaaatctct 1020ttggattctg
gtcatgagaa gaaggtaact tccagccttc aagaagacag acatttagaa
1080gaagagctga aatgtcagga acaaaaagaa gaacagctgc aggaaggggt
gcaccggaag 1140gagccccagg gggccacgta gcagcggctc agtgggtggc
catcgatctg gaccgtcccc 1200tgcccacttg ctccccgtga gcactgcgta
caaacatcca aaagttcaac aacaccagaa 1260ctgtgtgtct catggtatgt
aactcttaaa gcaaataaat gaactgactt caaaaaaaaa 1320a 1321162359DNAHomo
sapiens 16cccaaatgtc tcagaatgta tgtcccagaa acctgtggct gcttcaacca
ttgacagttt 60tgctgctgct ggcttctgca gacagtcaag ctgcagctcc cccaaaggct
gtgctgaaac 120ttgagccccc gtggatcaac gtgctccagg aggactctgt
gactctgaca tgccaggggg 180ctcgcagccc tgagagcgac tccattcagt
ggttccacaa tgggaatctc attcccaccc 240acacgcagcc cagctacagg
ttcaaggcca acaacaatga cagcggggag tacacgtgcc 300agactggcca
gaccagcctc agcgaccctg tgcatctgac tgtgctttcc gaatggctgg
360tgctccagac ccctcacctg gagttccagg agggagaaac catcatgctg
aggtgccaca 420gctggaagga caagcctctg gtcaaggtca cattcttcca
gaatggaaaa tcccagaaat 480tctcccgttt ggatcccacc ttctccatcc
cacaagcaaa ccacagtcac agtggtgatt 540accactgcac aggaaacata
ggctacacgc tgttctcatc caagcctgtg accatcactg 600tccaagtgcc
cagcatgggc agctcttcac caatggggat cattgtggct gtggtcattg
660cgactgctgt agcagccatt gttgctgctg tagtggcctt gatctactgc
aggaaaaagc 720ggatttcagc caattccact gatcctgtga aggctgccca
atttgagcca cctggacgtc 780aaatgattgc catcagaaag agacaacttg
aagaaaccaa caatgactat gaaacagctg 840acggcggcta catgactctg
aaccccaggg cacctactga cgatgataaa aacatctacc 900tgactcttcc
tcccaacgac catgtcaaca gtaataacta aagagtaacg ttatgccatg
960tggtcatact ctcagcttgc tgatggatga caaaaagagg ggaattgtta
aaggaaaatt 1020taaatggaga ctggaaaaat cctgagcaaa caaaaccacc
tggcccttag aaatagcttt 1080aactttgctt aaactacaaa cacaagcaaa
acttcacggg gtcatactac atacaagcat 1140aagcaaaact taacttggat
catttctggt aaatgcttat gttagaaata agacaacccc 1200agccaatcac
aagcagccta ctaacatata attaggtgac tagggacttt ctaagaagat
1260acctaccccc aaaaaacaat tatgtaattg aaaaccaacc gattgccttt
attttgcttc 1320cacattttcc caataaatac ttgcctgtga cattttgcca
ctggaacact aaacttcatg 1380aattgcgcct cagatttttc ctttaacatc
tttttttttt ttgacagagt ctcaatctgt 1440tacccaggct ggagtgcagt
ggtgctatct tggctcactg caaacccgcc tcccaggttt 1500aagcgattct
tatgcctcag cctcccagta gctgggatta gaggcatgtg ccatcatacc
1560cagctaattt ttgtattttt tattttttat ttttagtaga gacagggttt
cgcaatgttg 1620gccaggccga tctcgaactt ctggcctcta gcgatctgcc
cgcctcggcc tcccaaagtg 1680ctgggatgac cgcatcagcc ccaatgtcca
gcctctttaa catcttcttt cctatgccct 1740ctctgtggat ccctactgct
ggtttctgcc ttctccatgc tgagaacaaa atcacctatt 1800cactgcttat
gcagtcggaa gctccagaag aacaaagagc ccaattacca gaaccacatt
1860aagtctccat tgttttgcct tgggatttga gaagagaatt agagaggtga
ggatctggta 1920tttcctggac taaattccct tggggaagac gaagggatgc
tgcagttcca aaagagaagg 1980actcttccag agtcatctac ctgagtccca
aagctccctg tcctgaaagc cacagacaat 2040atggtcccaa atgactgact
gcaccttctg tgcctcagcc gttcttgaca tcaagaatct 2100tctgttccac
atccacacag ccaatacaat tagtcaaacc actgttatta acagatgtag
2160caacatgaga aacgcttatg ttacaggtta catgagagca atcatgtaag
tctatatgac 2220ttcagaaatg ttaaaataga ctaacctcta acaacaaatt
aaaagtgatt gtttcaaggt 2280gatgcaatta ttgatgacct attttatttt
tctataatga tcatatatta cctttgtaat 2340aaaacattat aaccaaaac
235917887DNAHomo sapiens 17tctttggtga cttgtccact ccagtgtggc
atcatgtggc agctgctcct cccaactgct 60ctgctacttc tagtttcagc tggcatgcgg
actgaagatc tcccaaaggc tgtggtgttc 120ctggagcctc aatggtacag
cgtgcttgag aaggacagtg tgactctgaa gtgccaggga 180gcctactccc
ctgaggacaa ttccacacag tggtttcaca atgagagcct catctcaagc
240caggcctcga gctacttcat tgacgctgcc acagtcaacg acagtggaga
gtacaggtgc 300cagacaaacc tctccaccct cagtgacccg gtgcagctag
aagtccatat cggctggctg 360ttgctccagg cccctcggtg ggtgttcaag
gaggaagacc ctattcacct gaggtgtcac 420agctggaaga acactgctct
gcataaggtc acatatttac agaatggcaa agacaggaag 480tattttcatc
ataattctga cttccacatt ccaaaagcca cactcaaaga tagcggctcc
540tacttctgca gggggcttgt tgggagtaaa aatgtgtctt cagagactgt
gaacatcacc 600atcactcaag gtttggcagt gtcaaccatc tcatcattct
ctccacctgg gtaccaagtc 660tctttctgct tggtgatggt actccttttt
gcagtggaca caggactata tttctctgtg 720aagacaaaca tttgaagctc
aacaagagac tggaaggacc ataaacttaa atggagaaag 780gaccctcaag
acaaatgacc cccatcccat gggagtaata agagcagtgg cagcagcatc
840tctgaacatt tctctggatt tgcaacccca tcatcctcag gcctctc
887181503DNAHomo sapiens 18ctcctgctta aacctctgtc tctgacggtc
cctgccaatc gctctggtcg accccaacac 60actaggagga cagacacagg ctccaaactc
cactaagtga ccagagctgt gattgtgccc 120gctgagtgga ctgcgttgtc
agggagtgag tgctccatca tcgggagaat ccaagcagga 180ccgccatgga
ggaaggtcaa tattcagaga tcgaggagct tcccaggagg cggtgttgca
240ggcgtgggac tcagatcgtg ctgctggggc tggtgaccgc cgctctgtgg
gctgggctgc 300tgactctgct tctcctgtgg cactgggaca ccacacagag
tctaaaacag ctggaagaga 360gggctgcccg gaacgtctct caagtttcca
agaacttgga aagccaccac ggtgaccaga 420tggcgcagaa atcccagtcc
acgcagattt cacaggaact ggaggaactt cgagctgaac 480agcagagatt
gaaatctcag gacttggagc tgtcctggaa cctgaacggg cttcaagcag
540atctgagcag cttcaagtcc caggaattga acgagaggaa cgaagcttca
gatttgctgg 600aaagactccg ggaggaggtg acaaagctaa ggatggagtt
gcaggtgtcc agcggctttg 660tgtgcaacac gtgccctgaa aagtggatca
atttccaacg gaagtgctac tacttcggca 720agggcaccaa gcagtgggtc
cacgcccggt atgcctgtga cgacatggaa gggcagctgg 780tcagcatcca
cagcccggag gagcaggact tcctgaccaa gcatgccagc cacaccggct
840cctggattgg ccttcggaac ttggacctga agggagagtt tatctgggtg
gatgggagcc 900atgtggacta cagcaactgg gctccagggg agcccaccag
ccggagccag ggcgaggact 960gcgtgatgat gcggggctcc ggtcgctgga
acgacgcctt ctgcgaccgt aagctgggcg 1020cctgggtgtg cgaccggctg
gccacatgca cgccgccagc cagcgaaggt tccgcggagt 1080ccatgggacc
tgattcaaga ccagaccctg acggccgcct gcccaccccc tctgcccctc
1140tccactcttg agcatggata cagccaggcc cagagcaaga ccctgaagac
ccccaaccac 1200ggcctaaaag cctctttgtg gctgaaaggt ccctgtgaca
ttttctgcca cccaaacgga 1260ggcagctgac acatctcccg ctcctctatg
gcccctgcct tcccaggagt acaccccaac 1320agcaccctct ccagatggga
gtgcccccaa cagcaccctc tccagatgag agtacacccc 1380aacagcaccc
tctccagatg cagccccatc tcctcagcac cccaggacct gagtatcccc
1440agctcaggtg gtgagtcctc ctgtccagcc tgcatcaata aaatggggca
gtgatggcct 1500ccc 15031932DNAArtificial sequencePrimer for
Fc(gamma)RIIb2 19aatagaattc catggggaca cctgcagctc cc
322028DNAArtificial sequencePrimer for Fc(gamma)RIIb2 20cccagtgtcg
acagcctaaa tgatcccc 28
* * * * *