U.S. patent application number 16/020531 was filed with the patent office on 2018-10-18 for diagnosis of a novel autoimmune disease.
This patent application is currently assigned to EUROIMMUN MEDIZINISCHE LABORDIAGNOSTIKA AG. The applicant listed for this patent is EUROIMMUN MEDIZINISCHE LABORDIAGNOSTIKA AG. Invention is credited to Yvonne Denno, Inga-Madeleine Dettmann, Lars Komorowski, Nico Melzer, Sven Meuth, Ramona Miske, Christian Probst, Madeleine Scharf, Winfried Stocker, Bianca Teegen, Heinz Wiendl.
Application Number | 20180298072 16/020531 |
Document ID | / |
Family ID | 53015464 |
Filed Date | 2018-10-18 |
United States Patent
Application |
20180298072 |
Kind Code |
A1 |
Miske; Ramona ; et
al. |
October 18, 2018 |
DIAGNOSIS OF A NOVEL AUTOIMMUNE DISEASE
Abstract
Polypeptides including Neurochondrin and autoantibodies binding
to polypeptides including Neurochondrin are provided. Methods for
diagnosing or treating diseases associated with neurological
symptoms or cancers are also provided. The methods of diagnosis may
include detecting an autoantibody binding to Neurochondrin in a
sample from a patient. The methods of treatment may include
administering a polypeptide comprising Neurochondrin to a
patient.
Inventors: |
Miske; Ramona; (Luebeck,
DE) ; Scharf; Madeleine; (Luebeck, DE) ;
Komorowski; Lars; (Ratzeburg, DE) ; Denno;
Yvonne; (Luebeck, DE) ; Probst; Christian;
(Ratzeburg, DE) ; Dettmann; Inga-Madeleine;
(Ahrensboek, DE) ; Stocker; Winfried; (Gross
Groenau, DE) ; Teegen; Bianca; (Rehna, DE) ;
Melzer; Nico; (Muenster, DE) ; Wiendl; Heinz;
(Muenster, DE) ; Meuth; Sven; (Muenster,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EUROIMMUN MEDIZINISCHE LABORDIAGNOSTIKA AG |
Luebeck |
|
DE |
|
|
Assignee: |
EUROIMMUN MEDIZINISCHE
LABORDIAGNOSTIKA AG
Luebeck
DE
|
Family ID: |
53015464 |
Appl. No.: |
16/020531 |
Filed: |
June 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15078361 |
Mar 23, 2016 |
|
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16020531 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/4713 20130101;
A61P 25/28 20180101; A61P 35/00 20180101; G01N 33/564 20130101;
A61P 25/14 20180101; G01N 33/6896 20130101; A61K 38/00 20130101;
A61P 25/16 20180101; G01N 2333/47 20130101; C07K 2317/14 20130101;
A61P 25/00 20180101; C07K 16/18 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; G01N 33/564 20060101 G01N033/564; C07K 16/18 20060101
C07K016/18; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2015 |
EP |
15001186.4 |
Claims
1-15. (canceled)
16. A method of detecting an autoantibody to Neurochondrin in a
subject, the method comprising: contacting a bodily fluid sample
isolated from a subject having cerebellar ataxia or cerebellitis
with a polypeptide comprising an amino acid sequence encoded by a
nucleic acid sequence that hybridizes under stringent conditions to
the nucleic acid sequence of SEQ ID NO:1, wherein the polypeptide
is selected from at least one of a recombinant polypeptide and an
isolated polypeptide; and detecting the presence or absence of the
autoantibody to Neurochondrin in a complex with the
polypeptide.
17. The method of claim 16, the stringent conditions comprising a
salt concentration and a washing temperature, wherein the salt
concentration is selected from between about 0.1.times.SSC to about
2.times.SSC and the washing temperature is selected from between
about 50.degree. C. to about 68.degree. C.
18. The method of claim 16, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, and SEQ ID NO:9.
19. The method of claim 16, wherein the polypeptide is immobilized
on a solid carrier.
20. The method of claim 19, wherein the solid carrier is selected
from at least one of glass plates, slides, biochips, microtiter
plates, magnetic beads, chromatography columns, and membranes.
21. The method of claim 16, wherein the polypeptide is provided in
the form of an isolated cell comprising a nucleic acid encoding the
polypeptide or in the form of an isolated tissue comprising the
polypeptide.
22. The method of claim 16, wherein the subject is at least one of
a human and a mammal.
23. The method of claim 16, wherein the detecting the presence or
absence of the autoantibody to Neurochondrin in a complex with the
polypeptide comprises the use of a secondary antibody.
24. The method of claim 16, wherein the detecting the presence or
absence of the autoantibody to Neurochondrin in a complex with the
polypeptide comprises the use of at least one immunoassay technique
selected from radioimmunoassay, western blot, line blot,
enzyme-linked immunosorbent assay (ELISA), direct
immunofluorescence, and indirect immunofluorescence.
25. The method of claim 16, wherein the bodily fluid is selected
from the group comprising cerebrospinal fluid, whole blood, blood
serum, lymph, interstitial fluid, and saliva.
26. A method of detecting an autoantibody to Neurochondrin in a
subject, the method comprising: contacting a bodily fluid sample
isolated from a subject having cerebellar ataxia or cerebellitis
with a polypeptide comprising Neurochondrin or a variant thereof,
wherein the polypeptide is selected from at least one of a
recombinant polypeptide and an isolated polypeptide; and detecting
the presence or absence of the autoantibody to Neurochondrin in a
complex with the polypeptide.
27. The method of claim 26, wherein the variant comprises an amino
acid sequence having at least about 90% sequence identity to
Neurochondrin.
28. The method of claim 26, wherein the variant comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, and SEQ ID NO:9.
29. The method of claim 26, wherein the polypeptide is immobilized
on a solid carrier.
30. The method of claim 29, wherein the solid carrier is selected
from at least one of glass plates, slides, biochips, microtiter
plates, magnetic beads, chromatography columns, and membranes.
31. The method of claim 26, wherein the polypeptide is provided in
the form of an isolated cell comprising a nucleic acid encoding the
polypeptide or in the form of an isolated tissue comprising the
polypeptide.
32. The method of claim 26, wherein the subject is at least one of
a human and a mammal.
33. The method of claim 26, wherein the detecting the presence or
absence of the autoantibody to Neurochondrin in a complex with the
polypeptide comprises the use of a secondary antibody.
34. The method of claim 26, wherein the detecting the presence or
absence of the autoantibody to Neurochondrin in a complex with the
polypeptide comprises the use of at least one immunoassay technique
selected from radioimmunoassay, western blot, line blot,
enzyme-linked immunosorbent assay (ELISA), direct
immunofluorescence, and indirect immunofluorescence.
35. The method of claim 26, wherein the bodily fluid is selected
from the group comprising cerebrospinal fluid, whole blood, blood
serum, lymph, interstitial fluid, and saliva.
36. A method of detecting a complex comprising an autoantibody
bound to a polypeptide, the method comprising: contacting a bodily
fluid sample isolated from a subject having cerebellar ataxia or
cerebellitis with a polypeptide comprising an amino acid sequence
encoded by a nucleic acid sequence that hybridizes under stringent
conditions to the nucleic acid sequence of SEQ ID NO:1, wherein the
polypeptide is selected from at least one of a recombinant
polypeptide and an isolated polypeptide, and detecting whether at
least one complex comprising the autoantibody bound to the
polypeptide is present in the bodily fluid sample.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of European Patent
Application No. 15001186.4, filed Apr. 22, 2015, which is hereby
incorporated by reference in its entirety.
SUMMARY
[0002] The present disclosure relates to methods for diagnosing a
disease, for example, a disease associated with neurological
symptoms and/or a cancer, comprising the step of detecting in a
sample from a patient an autoantibody binding to Neurochondrin;
polypeptides comprising Neurochondrin or variants thereof, which
are immobilized, in some embodiments, on a solid carrier; uses of a
polypeptide comprising Neurochondrin or a variant thereof for the
diagnosis of a disease, in certain embodiments, comprising the step
of detecting autoantibodies binding to Neurochondrin; polypeptides
comprising Neurochondrin or variants thereof, in various
embodiments, immobilized, in some embodiments, on a solid carrier,
for use in the treatment of a disease; autoantibodies, in certain
embodiments, an isolated autoantibody, binding to a polypeptide
comprising Neurochondrin, wherein, in various embodiments, the
autoantibody is in complex with said polypeptide or a variant
thereof; methods for isolating an autoantibody binding to
Neurochondrin, comprising the steps of: a) contacting a sample
comprising the autoantibody with a polypeptide comprising
Neurochondrin or a variant thereof under conditions compatible with
formation of a complex, wherein said autoantibody binds to said
polypeptide; b) isolating the complex formed in step a); c)
dissociating the complex isolated in step b); and d) separating the
autoantibody from the polypeptide; pharmaceutical compositions
comprising a polypeptide comprising Neurochondrin or a variant
thereof; medical or diagnostic devices comprising a polypeptide
comprising Neurochondrin or a variant thereof; and test kits for
the diagnosis of a disease, comprising a polypeptide comprising
Neurochondrin or a variant thereof, wherein, in some embodiments,
the test kit comprises, in addition, a means for detecting the
complex comprising an autoantibody binding to Neurochondrin.
DETAILED DESCRIPTION
[0003] Developing diagnostic systems for neurological diseases is a
continuing challenge in biomedical science, not in the least
because many symptoms encountered may be accounted for by a huge
variety of causes including genetically-inherited diseases, drug
abuse, malnutrition, infection, injury, psychiatric illness,
immunological defects and cancer.
[0004] Since a neurological disease is rarely associated with a
unique characteristic pattern of clinical symptoms, it is often
difficult to provide a reliable diagnosis solely based on the
observation and examination of the patients affected or their
medical history.
[0005] The importance of an early diagnosis cannot be
overemphasized. Many neurological disorders, most prominently
Alzheimer's and Parkinson's diseases, cannot be cured, but drugs
are available that may be used to slow down their progression. The
earlier the diagnosis, the better the chances to exploit the
spectrum of available drugs to the full benefit of the patient.
[0006] This holds all the more true in the case of neurological
diseases associated with autoantibodies. In some cases, the link
between a specific detectable autoantibody and a condition is
sufficiently strong to allow for an immediate diagnosis.
[0007] But even if it is not, the detection of autoantibodies may
point the physician in charge to therapeutic means that may be used
to ameliorate the patient's condition. There is a variety of widely
used immunosuppressants that may be used regardless of the nature
of the autoantibody's target. Alternatively, apheresis may be used
to remove autoantibodies from the patient's blood. In many cases,
patients went on to lead a normal life following early diagnosis
and treatment of a neurological autoimmune disease.
[0008] Diagnostic assays based on the detection of autoantibodies
may also corroborate the diagnosis of diseases other than those
associated with autoantibodies. If it turns out that a blood sample
is devoid of specific autoantibodies, this is likely to help the
physician in charge exclude a range of possibilities and thus
narrow down the spectrum of plausible conditions.
[0009] Examples of neurological conditions coinciding with the
emergence of autoantibodies include Neuromyelitis optica, a disease
characterized by loss of vision and spinal cord function, and
anti-NMDA receptor encephalitis, which is associated with autonomic
dysfunction, hypoventilation, cerebellar ataxia, hemiparesis, loss
of consciousness, or catatonia. Whilst the involvement of
autoantibodies and the nature of these conditions as such were
previously poorly understood, many of these diseases can now be
diagnosed and treated efficiently owing to the availability of
assays based on the detection of autoantibodies.
[0010] Therefore, it is paramount that new approaches to
distinguish neurological conditions associated with autoantibodies
from those that are not be developed.
[0011] The problem underlying the present disclosure is to provide
novel reagents, devices and methods that may be used to support the
diagnosis and treatment of a neurological disease, in particular a
neurological disease associated with one or more symptoms from the
group comprising cerebrellar oculomotor disturbances, dysarthria,
ataxia, asthenia, asynergy, delayed reaction time, dyschronometria,
instability of gait, difficulty with eye movements, dysphagia,
hypotonia, inflammatory cerebrospinal fluid changes, dysmetria and
dydiadochokinesia.
[0012] Another problem underlying the present disclosure is to
provide novel reagents, devices and methods that may be used to
distinguish autoimmune diseases, in particular neurological
autoimmune diseases, from diseases other than autoimmune diseases,
not in the least for the purpose of determining the most promising
treatment regimen for the patient affected, more specifically
whether or not an immunosuppressive treatment is adequate.
[0013] The problem underlying the present disclosure is solved by
the subject-matter of the attached independent and dependent
claims.
[0014] In a first aspect, the problem underlying the present
disclosure is solved by a method for diagnosing a disease, for
example, a disease associated with neurological symptoms and/or a
tumor, comprising the step of detecting in a sample from a patient
an autoantibody binding to Neurochondrin.
[0015] In an embodiment of the first aspect, the sample is a bodily
fluid comprising antibodies, for example, selected from the group
comprising whole-blood, serum, cerebrospinal fluid and saliva.
[0016] In a second aspect, the problem underlying the present
disclosure is solved by a polypeptide comprising Neurochondrin or a
variant thereof, which is immobilized, for example, on a solid
carrier.
[0017] In a third aspect, the problem underlying the present
disclosure is solved by a use of a polypeptide comprising
Neurochondrin or a variant thereof for the diagnosis of a disease,
for example, comprising the step of detecting autoantibodies
binding to Neurochondrin.
[0018] In a fourth aspect, the problem underlying the present
disclosure is solved by a polypeptide comprising Neurochondrin or a
variant thereof, for example, immobilized, for example, on a solid
carrier, for use in the treatment of a disease.
[0019] In a fifth aspect, the problem underlying the present
disclosure is solved by an autoantibody, for example, an isolated
autoantibody, binding to a polypeptide comprising Neurochondrin,
wherein the autoantibody is, for example, in complex with said
polypeptide or a variant thereof.
[0020] In a sixth aspect, the problem underlying the present
disclosure is solved by a method for isolating an autoantibody
binding to Neurochondrin, comprising the steps of: a) contacting a
sample comprising the autoantibody with a polypeptide comprising
Neurochondrin or a variant thereof under conditions compatible with
formation of a complex, wherein said autoantibody binds to said
polypeptide; b) isolating the complex formed in step a); c)
dissociating the complex isolated in step b); and d) separating the
autoantibody from the polypeptide.
[0021] In a seventh aspect, the problem underlying the present
disclosure is solved by a pharmaceutical composition comprising a
polypeptide comprising Neurochondrin or a variant thereof.
[0022] In an eighth aspect, the problem underlying the present
disclosure is solved by a medical or diagnostic device comprising a
polypeptide comprising Neurochondrin or a variant thereof.
[0023] In an ninth aspect, the problem underlying the present
disclosure is solved by a test kit for the diagnosis of a disease,
for example, a disease associated with neurological symptoms and/or
a cancer, which test kit comprises a polypeptide comprising
Neurochondrin or a variant thereof, wherein, for example, the test
kit comprises, in addition, a means for detecting the complex
comprising an autoantibody binding to Neurochondrin or a variant
thereof. In certain embodiments, the test kit is configured to
detect the presence or absence of at least one complex comprising
an autoantibody bound to (i) Neurochondrin or a variant thereof or
(ii) a polypeptide comprising Neurochondrin or a variant
thereof.
[0024] In an embodiment of any aspect or embodiment of the
disclosure, the patient has or the disease is associated with one
or more symptoms from the group comprising cerebrellar oculomotor
disturbances, dysarthria, ataxia, asthenia, asynergy, delayed
reaction time, dyschronometria, instability of gait, difficulty
with eye movements, dysphagia, hypotonia, inflammatory
cerebrospinal fluid changes, dysmetria and dydiadochokinesia,
optionally further comprising cerebellar signal alterations,
brainstem signal alterations, atrophy on magnetic resonance imaging
and a cancer.
[0025] In an embodiment of any aspect or embodiment of the
disclosure, the patient has or the disease is associated with one
or more symptoms from the group comprising cerebrellar oculomotor
disturbances, dysarthria and ataxia.
[0026] In an embodiment of any aspect or embodiment of the
disclosure, the disease is associated with neurological or
psychiatric symptoms and is, for example, a neurological autoimmune
disease.
[0027] In an embodiment of any aspect or embodiment of the
disclosure, the polypeptide is provided in the form of a cell
comprising a nucleic acid encoding said polypeptide or in the form
of a tissue comprising said polypeptide.
[0028] In an embodiment of any aspect or embodiment of the
disclosure, the polypeptide is a recombinant and/or isolated
polypeptide.
[0029] The present disclosure is based on the inventors' surprising
finding that a neurological autoimmune disease exists that is
associated with autoantibodies to Neurochondrin (NCDN) and symptoms
from the group comprising cerebrellar oculomotor disturbances,
dysarthria, ataxia, asthenia, asynergy, delayed reaction time,
dyschronometria, instability of gait, difficulty with eye
movements, dysphagia, hypotonia, inflammatory cerebrospinal fluid
changes, dysmetria and dydiadochokinesia.
[0030] Furthermore, the present disclosure is based on the
inventors' surprising finding that autoantibodies to NCDN exist and
may be detected in samples from a number of patients suffering from
neurological symptoms, but not in samples obtained from healthy
subjects. Without wishing to be bound to any theory, the presence
of such autoantibodies suggests that activity and function NCDN
and/or downstream effectors is impaired in patients having NCDN
autoantibodies to the effect that neurological symptoms occur.
[0031] NCDN is a 75 k Da protein which is evolutionarily conserved
from invertebrates to vertebrates. The orthologous rat, murine and
human proteins have an amino acid identity of 98%.
[0032] NCDN is rich in .alpha.-helices (65%) which are organized as
tandem repeats. The protein was found to interact with several
membrane proteins from the brain, including Sema4C, MCHR1 and GRM5.
These interactions involve the C-terminal part of NCDN and the
membrane-proximal region of the respective membrane proteins
(Francke F., Ward R. J., Jenkins L., Kellett E., Richter D.,
Milligan G. and Bachner D., 2006, Interaction of neurochondrin with
the melanin-concentrating hormone receptor 1 interferes with G
protein-coupled signal transduction but not agonist-mediated
internalization, The Journal of biological chemistry, 281, 43,
32496-32507; Wang H., Westin L., Nong Y., Birnbaum S., Bendor J.,
Brismar H., Nestler E., Aperia A., Flajolet M. and Greengard P.,
2009, Norbin is an endogenous regulator of metabotropic glutamate
receptor 5 signaling, Science (New York, N.Y.), 326, 5959,
1554-1557).
[0033] NCDN was found to induce neurite outgrowth when ectopically
overexpressed in cultured neuroblastoma N2a cells. It was shown
that NCDN recruits Dia, an actin nucleation factor that stimulates
barbed end actin filament elongation to a specific sub-cellular
localization. This recruitment contributes to neurite-outgrowth
function of NCDN (Schwaibold E. M. and Brandt D. T., 2008,
Identification of Neurochondrin as a new interaction partner of the
FH3 domain of the Diaphanous-related formin Dia1, Biochem Biophys
Res Commun, 373, 3, 366-372).
[0034] NCDN modulates the signaling pathway of GRM5 (Wang H.,
Westin L., Nong Y., Birnbaum S., Bendor J., Brismar H., Nestler E.,
Aperia A., Flajolet M. and Greengard P., 2009, Norbin is an
endogenous regulator of metabotropic glutamate receptor 5
signaling, Science (New York, N.Y.), 326, 5959, 1554-1557). GRM5
primarily responds to the excitatory amino acid neurotransmitter
glutamate, localizes at perisynaptic sites and plays important
roles in normal brain function as well as in several pathological
disorders including schizophrenia (Abe T., Sugihara H., Nawa H.,
Shigemoto R., Mizuno N. and Nakanishi S., 1992, Molecular
characterization of a novel metabotropic glutamate receptor mGluR5
coupled to inositol phosphate/Ca2+ signal transduction, The Journal
of biological chemistry, 267, 19, 13361-13368, Romano C., Sesma M.
A., McDonald C. T., O'Malley K., Van den Pol A. N. and Olney J. W.,
1995, Distribution of metabotropic glutamate receptor mGluR5
immunoreactivity in rat brain, The Journal of comparative
neurology, 355, 3, 455-469, Nakanishi S., Nakajima Y., Masu M.,
Ueda Y., Nakahara K., Watanabe D., Yamaguchi S., Kawabata S. and
Okada M., 1998, Glutamate receptors: brain function and signal
transduction, Brain research Brain research reviews, 26, 2-3,
230-235, Conn P. J., Christopoulos A. and Lindsley C. W., 2009,
Allosteric modulators of GPCRs: a novel approach for the treatment
of CNS disorders, Nature reviews Drug discovery, 8, 1, 41-54). NCDN
expression enhances GRM5 signaling in an in vitro expression
system, while the conditional knockout of NCDN in transgenic mice
reduces GRM5 function. In line with reduced GRM5 function, NCND
knockout mice show defects in pre-pulse inhibition and
psychostimulant induced locomotor activity, two behavioral
phenotypes typically observed in rodent models of schizophrenia
(Wang H., Westin L., Nong Y., Birnbaum S., Bendor J., Brismar H.,
Nestler E., Aperia A., Flajolet M. and Greengard P., 2009, Norbin
is an endogenous regulator of metabotropic glutamate receptor 5
signaling, Science (New York, N.Y.), 326, 5959, 1554-1557).
[0035] NCDN has been shown to inhibit MCHR-induced G-protein
activation and downstream calcium influx. MCHR1 is the
melanin-concentrating hormone receptor involved in the regulation
of feeding behavior and energy balances (Lembo P. M., Grazzini E.,
Cao J., Hubatsch D. A., Pelletier M., Hoffert C., St-Onge S., Pou
C., Labrecque J., Groblewski T., O'Donnell D., Payza K., Ahmad S.
and Walker P., 1999, The receptor for the orexigenic peptide
melanin-concentrating hormone is a G-protein-coupled receptor,
Nature Cell biology, 1, 5, 267-271).
[0036] NCDN is a negative regulator of Ca2+/calmodulin-dependent
protein kinase II (CaMKII) (Thr286) phosphorylation and essential
for the spatial learning process but not for the differentiation or
neurite outgrowth of the neuron. In addition, nervous
system-specific homozygous gene disruption resulted in epileptic
seizure (Dateki M., Horii T., Kasuya Y., Mochizuki R., Nagao Y.,
Ishida J., Sugiyama F., Tanimoto K., Yagami K., Imai H. and
Fukamizu A., 2005, Neurochondrin negatively regulates CaMKII
phosphorylation, and nervous system-specific gene disruption
results in epileptic seizure, The Journal of biological chemistry,
280, 21, 20503-20508).
[0037] The present disclosure relates to a polypeptide comprising a
mammalian, for example, human NCDN or variants thereof, for
example, immunogenic variants binding to NCDN autoantibodies. In an
embodiment, the polypeptide comprises NCDN as encoded by the data
base code Q9UBB6 or a variant thereof. Throughout this application,
any data base codes cited refer to the Uniprot data base, more
specifically the version accessible on-line on Apr. 17, 2015. SEQ
ID NO:1 represents a nucleotide sequence encoding NCDN. In an
embodiment, the polypeptide comprises an amino acid sequence
selected from the group comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ
ID NO:9.
[0038] The teachings of the present disclosure may not only be
carried out using polypeptides, in particular a polypeptide
comprising the native sequence of NCDN, or nucleic acids having the
exact sequences referred to in this application explicitly, for
example, by function, name, sequence or accession number, or
implicitly, but also using variants of such polypeptides or nucleic
acids.
[0039] In an embodiment, the term "variant," as used herein, may
refer to at least one fragment of the full length sequence referred
to, more specifically one or more amino acid or nucleic acid
sequence which is, relative to the full-length sequence, truncated
at one or both termini by one or more amino acids. Such a fragment
comprises or encodes for a peptide having at least 6, 7, 8, 10, 12,
15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids of the
original sequence or a variant thereof. The total length of the
variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50,
60, 70, 80, 90, 100 or more amino acids.
[0040] In another embodiment, the term "variant" relates not only
to at least one fragment, but also to a polypeptide or a fragment
thereof comprising amino acid sequences that are at least 40, 50,
60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to
the reference amino acid sequence referred to or the fragment
thereof, wherein amino acids other than those essential for the
biological activity, for example, the ability of an antigen to bind
to an (auto)antibody, or the fold or structure of the polypeptide
are deleted or substituted and/or one or more such essential amino
acids are replaced in a conservative manner and/or amino acids are
added such that the biological activity of the polypeptide is
preserved. The state of the art comprises various methods that may
be used to align two given nucleic acid or amino acid sequences and
to calculate the degree of identity, see, for example, Arthur Lesk
(2008), Introduction to bioinformatics, Oxford University Press,
2008, 3rd edition. In an embodiment, the ClustalW software (Larkin,
M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P.
A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez,
R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007). Clustal
W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used
using default settings.
[0041] In an embodiment, variants may, in addition, comprise
chemical modifications, for example, isotopic labels or covalent
modifications such as glycosylation, phosphorylation, acetylation,
decarboxylation, citrullination, hydroxylation and the like. The
person skilled in the art is familiar with methods to modify
polypeptides. Any modification is designed such that it does not
abolish the biological activity of the variant.
[0042] Moreover, variants may also be generated by fusion with
other known polypeptides or variants thereof and comprise active
portions or domains, for example, having a sequence identity of at
least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% when
aligned with the active portion of the reference sequence, wherein
the term "active portion," as used herein, refers to an amino acid
sequence, which is less than the full length amino acid sequence
or, in the case of a nucleic acid sequence, codes for less than the
full length amino acid sequence, respectively, and/or is a variant
of the natural sequence, but retains at least some of the
biological activity.
[0043] In an embodiment, the term "variant" of a nucleic acid
comprises nucleic acids the complementary strand of which
hybridizes, for example, under stringent conditions, to the
reference or wild type nucleic acid. Stringency of hybridization
reactions is readily determinable by one of ordinary skilled in the
art, and in general is an empirical calculation dependent on probe
length, washing temperature and salt concentration. In general,
longer probes require higher temperatures for proper annealing,
while shorter probes less so. Hybridization generally depends on
the ability of denatured DNA to reanneal to complementary strands
present in an environment below their melting temperature: the
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
may be used. As a result, higher relative temperatures would tend
to make the reaction conditions more stringent, while lower
temperature less so. For additional details and explanation of
stringency of hybridization reactions, see Ausubel, F. M. (1995),
Current Protocols in Molecular Biology. John Wiley & Sons, Inc.
Moreover, the person skilled in the art may follow the instructions
given in the manual Boehringer Mannheim GmbH (1993) The DIG System
Users Guide for Filter Hybridization, Boehringer Mannheim GmbH,
Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and
Schleifer, K. H. (1991) International Journal of Systematic
Bacteriology 41: 255-260 on how to identify DNA sequences by means
of hybridization. In an embodiment, stringent conditions are
applied for any hybridization, i.e., hybridization occurs only if
the probe is 70% or more identical to the target sequence. Probes
having a lower degree of identity with respect to the target
sequence may hybridize, but such hybrids are unstable and will be
removed in a washing step under stringent conditions, for example,
lowering the concentration of salt to 2.times.SSC or, optionally
and subsequently, to 0.5.times.SSC, while the temperature is, for
example, approximately 50.degree. C.-68.degree. C., approximately
52.degree. C.-68.degree. C., approximately 54.degree. C.-68.degree.
C., approximately 56.degree. C.-68.degree. C., approximately
58.degree. C.-68.degree. C., approximately 60.degree. C.-68.degree.
C., approximately 62.degree. C.-68.degree. C., approximately
64.degree. C.-68.degree. C., approximately 66.degree. C.-68.degree.
C. In an embodiment, the temperature is approximately 64.degree.
C.-68.degree. C. or approximately 66.degree. C.-68.degree. C. It is
possible to adjust the concentration of salt to 0.2.times.SSC or
even 0.1.times.SSC. Nucleic acid sequences having a degree of
identity with respect to the reference or wild type sequence of at
least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% may be
isolated. In an embodiment, the term variant of a nucleic acid
sequence, as used herein, refers to any nucleic acid sequence that
encodes the same amino acid sequence and variants thereof as the
reference nucleic acid sequence, in line with the degeneracy of the
genetic code.
[0044] The variant of the polypeptide has biological activity. In
an embodiment, such biological activity is the ability to bind
specifically to the NCDN autoantibodies found in patients suffering
from the disease identified by the inventors.
[0045] The disclosed polypeptide, which comprises NCDN or a variant
thereof, when used to carry out the teachings of the present
disclosure, may be provided in any form and at any degree of
purification, from tissues or cells comprising said polypeptide in
an endogenous form, for example, cells overexpressing the
polypeptide, crude or enriched lysates of such cells, to purified
and/or isolated polypeptide which is essentially pure. In an
embodiment, the polypeptide is a native polypeptide, wherein the
term "native polypeptide," as used herein, refers to a folded
polypeptide, for example, to a folded polypeptide purified from
tissues or cells, for example, from mammalian cells or tissues,
optionally from non-recombinant tissues or cells. In another
embodiment, the polypeptide is a recombinant protein, wherein the
term "recombinant," as used herein, refers to a polypeptide
produced using genetic engineering approaches at any stage of the
production process, for example, by fusing a nucleic acid encoding
the polypeptide to a strong promoter for overexpression in cells or
tissues or by engineering the sequence of the polypeptide itself.
The person skilled in the art is familiar with methods for
engineering nucleic acids and polypeptides encoded (for example,
described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989),
Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning--an
introduction, Chapman & Hall) and for producing and purifying
native or recombinant polypeptides (for example, Handbooks
"Strategies for Protein Purification," "Antibody Purification,"
"Purifying Challenging Proteins," "Recombinant Protein
Purification," "Affinity Chromatography," "Ion Exchange
Chromatography," "Gel Filtration (Size Exclusion Chromatography),"
"Hydrophobic Interaction Chromatography," "Multimodal
Chromatography" (2009/2010), published by GE Healthcare Life
Sciences, and in Burgess, R. R., Deutscher, M. P. (2009), Guide to
Protein Purification). In an embodiment, a polypeptide is pure if
at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in the
respective sample consists of said polypeptide as judged by SDS
polyacrylamide gel electrophoresis followed by Coomassie blue
staining and visual inspection.
[0046] If the disclosed polypeptide comprising NCDN or a variant
thereof is provided in the form of tissue, in some embodiments, the
tissue is mammalian tissue, for example, human, rat, primate,
donkey, mouse, goat, horse, sheep, pig or cow, for example, brain
tissue. If a cell lysate is used, in some embodiments, the cell
lysate comprises the membranes associated with the surface of the
cell. If said polypeptide is provided in the form of a recombinant
cell, in some embodiments, the recombinant cell is a eukaryotic
cell such as a yeast cell, for example, a cell from a multicellular
eukaryote such as a plant, mammal, frog or insect, for example,
from a mammal, for example, rat, human, primate, donkey, mouse,
goat, horse, sheep, pig or cow. For example, the cell may be a
HEK293 cell transfected with a nucleic acid functionally encoding
the disclosed polypeptide. The person skilled in the art is
familiar with methods for preparing, transfecting and culturing
such cells, for example, those described in Phelan, M. C. (2001),
Basic Techniques in Mammalian Cell Tissue Culture, John Wiley.
[0047] The polypeptide used to carry out the disclosed teachings,
including any variants, may be designed such that it comprises
epitopes recognized by and/or binds specifically to autoantibodies
binding to NCDN. In one embodiment, such polypeptide comprises a
stretch of 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80,
90, 100 or more, for example, at least 9 but no more than 16,
consecutive amino acids from NCDN. The person skilled in the art is
familiar with guidelines used to design peptides having sufficient
immunogenicity, for example, those described in Jackson, D. C.,
Fitzmaurice, C. J., Brown, L. E., Zeng, W. (1999), Preparation and
properties of totally synthetic immunogenes, Vaccine Volume 18,
Issues 3-4, September 1999, Pages 355-361; and Black, M., Trent,
A., Tirrell, M. and Olive, C. (2010), Advances in the design and
delivery of peptide subunit vaccines with a focus on Toll-like
receptor agonists, Expert Rev Vaccines, 2010 February; 9(2):
157-173. Briefly, it is desirable that the peptide meets as many as
possible of the following requirements: (a) it has a high degree of
hydrophilicity, (b) it comprises one or more residues selected from
the group comprising aspartate, proline, tyrosine and
phenylalanine, (c) it has, for higher specificity, no or little
homology with other known peptides or polypeptides, (d) it needs to
be sufficiently soluble and (e) it comprises no glycosylation or
phosphorylation sites unless required for specific reasons.
Alternatively, bioinformatics approaches may be followed, for
example, those described by Moreau, V., Fleury, C., Piquer, D.,
Nguyen, C., Novali, N., Villard, S., Laune, D., Granier, C. and
Molina, F. (2008), PEPOP: Computational design of immunogenic
peptides, BMC Bioinformatics 2008, 9:71.
[0048] The disclosed polypeptide, which comprises NCDN or a variant
thereof, when used according to the present disclosure, may be
provided in any kind of conformation. For example, the polypeptide
may be an essentially unfolded, a partially or a fully folded
polypeptide. In an embodiment, the polypeptide is folded in the
sense that the epitopes essential for the binding to the disclosed
autoantibody, or the protein or variant thereof in its entirety,
adopt the fold adopted by the native protein in its natural
environment. The person skilled in the art is familiar with methods
suitable to determine whether or not a polypeptide is folded and if
it is, which structure it has, for example, limited proteolysis,
NMR spectroscopy, CD spectroscopy or X-ray crystallography (see,
for example, Banaszak L. J. (2008), Foundations of Structural
Biology, Academics Press, or Teng Q. (2013), Structural Biology:
Practical Applications, Springer), for example, multidimensional
NMR spectroscopy is used.
[0049] The disclosed polypeptide may be a fusion protein which
comprises amino acid sequences other than those taken from NCDN, in
particular a C-terminal or N-terminal tag, for example, a
C-terminal tag, which is, in an embodiment, as used herein, an
additional sequence motif or polypeptide having a function that has
some biological or physical function and may, for example, be used
to purify, immobilize, precipitate or identify the disclosed
polypeptide. In an embodiment, the tag is a sequence or domain
capable of binding specifically to a ligand, for example, a tag
selected from the group comprising His tags, thioredoxin, maltose
binding protein, glutathione-S-transferase, a fluorescence tag, for
example, from the group comprising green fluorescent protein.
[0050] The disclosed polypeptide may be an immobilized polypeptide.
In an embodiment, the term "immobilized," as used herein, refers to
a molecule bound to a solid carrier insoluble in an aqueous
solution, for example, via a covalent bond, electrostatic
interactions, encapsulation or entrapment, for example, by
denaturing a globular polypeptide in a gel, or via hydrophobic
interactions, for example, via one or more covalent bonds. Various
suitable carriers, for example, paper, polystyrene, metal, silicon
or glass surfaces, microfluidic channels, membranes, beads such as
magnetic beads, column chromatography media, biochips,
polyacrylamide gels and the like have been described in the
literature, for example, in Kim, D., and Herr, A. E. (2013),
Protein immobilization techniques for microfluidic assays,
Biomicrofluidics 7(4), 041501. This way, the immobilized molecule,
together with the insoluble carrier, may be separated from an
aqueous solution in a straightforward manner, for example, by
filtration, centrifugation or decanting. An immobilized molecule
may be immobilized in a reversible or irreversible manner. For
example, the immobilization is reversible if the molecule interacts
with the carrier via ionic interactions that can be masked by
addition of a high concentration of salt or if the molecule is
bound via a cleavable covalent bond such as a disulphide bridge
which may be cleaved by addition of thiol-containing reagents. By
contrast, the immobilization is irreversible if the molecule is
tethered to the carrier via a covalent bond that cannot be cleaved
in aqueous solution, for example, a bond formed by reaction of an
epoxide group and an amine group as frequently used to couple
lysine side chains to affinity columns. The protein may be
indirectly immobilized, for example, by immobilizing an antibody or
other entity having affinity to the molecule, followed by formation
of a complex to the effect that the molecule-antibody complex is
immobilized. Various ways to immobilize molecules are described in
the literature, for example, in Kim, D., Herr, and A. E. (2013),
Protein immobilization techniques for microfluidic assays,
Biomicrofluidics 7(4), 041501. In addition, various reagents and
kits for immobilization reactions are commercially available, for
example, from Pierce Biotechnology.
[0051] It is essential that the sample used for the diagnosis in
line with the present disclosure comprises antibodies, also
referred to as immunglobulins. Typically the sample of a bodily
fluid comprises a representative set of the entirety of the
subject's immunglobulins. However, the sample, once provided, may
be subjected to further processing which may include fractionation,
centrifugation, enriching or isolating the entirety of
immunglobulins or any immunglobulin class of the subject, which may
affect the relative distribution of immunglobulins of the various
classes.
[0052] The reagents, devices, methods and uses described throughout
this application may be used for the diagnosis of a disease. In an
embodiment, the disease is a neurological disease. In an
embodiment, the term "neurological disease," as used herein, refers
to any disease associated with a defect of the nervous system, for
example, the central nervous system, for example, the brain.
[0053] In an embodiment, the disease is a disease, for example, a
neurological disease, associated with one or more symptoms, for
example, two or more, for example, three or more from the group
comprising cerebrellar oculomotor disturbances, dysarthria, ataxia,
asthenia, asynergy, delayed reaction time, dyschronometria,
instability of gait, difficulty with eye movements, dysphagia,
hypotonia, inflammatory cerebrospinal fluid changes, dysmetria and
dydiadochokinesia, optionally further comprising cerebellar signal
alterations, brainstem signal alterations, atrophy on magnetic
resonance imaging and a tumor/cancer.
[0054] In another embodiment, the disease is a neurological disease
selected from the group comprising Alzheimer's Disease, Autism,
Asperger's Syndrome, Apraxia, Aphasia, Cerebellar syndrome,
Cerebellitis, Chorea, Encephalitis, Movement disorder,
spinocerebellar ataxia, for example, a non-progressive form,
Paralysis, Paraplegia, Gaucher's disease, Myopathy, Myasthenia
gravis, Multiple Sclerosis, Parkinsons's disease, Polyneuropathy
and Dementia, for example, Cerebellar syndrome, Cerebellitis,
Movement disorder and Dementia.
[0055] In another embodiment, the disease is a cancer or, for
example, paraneoplastic neurological syndrome, which is associated
both with one or more neurological symptoms, for example, from the
group comprising cerebrellar oculomotor disturbances, dysarthria,
ataxia, asthenia, asynergy, delayed reaction time, dyschronometria,
instability of gait, difficulty with eye movements, dysphagia,
hypotonia, inflammatory cerebrospinal fluid changes, dysmetria and
dydiadochokinesia, optionally further comprising cerebellar signal
alterations, brainstem signal alterations, and atrophy on magnetic
resonance imaging and is furthermore associated with a cancer.
Detection of autoantibodies to Neurochrondrin may indicate an
increased likelihood that a cancer is present which cannot be
detected using other methods, or will appear as the disease
progresses. In an embodiment, the cancer is a cancer of tumor
selected from the group comprising tumor of the lung, tumor of the
thymus, thymic tumor, testicular tumor, head and neck cancer tumor,
breast cancer tumor, ano-genital cancer tumor, melanoma, sarcoma,
carcinoma, lymphoma, leukemia, mesothelioma, glioma, germ cell
tumor, choriocarcinoma, pancreatic cancer, ovarian cancer, gastric
cancer, carcinomatous lesion of the pancreas, pulmonary
adenocarcinoma, colorectal adenocarcinoma, pulmonary squamous
adenocarcinoma, gastric adenocarcinoma, ovarian surface epithelial
neoplasm (e.g., a benign, proliferative or malignant variety
thereof), oral squamous cell carcinoma, non small-cell lung
carcinoma, endometrial carcinoma, a bladder cancer, prostate
carcinoma, acute myelogenous leukemia (AML), myelodysplasia
syndrome (MDS), non-small cell lung cancer (NSCLC), Wilms' tumor,
leukemia, lymphoma, desmoplastic small round cell tumor,
mesothelioma (e.g., malignant mesothelioma), a gastric cancer,
colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian
cancer, uterine cancer, thyroid cancer, hepatocellular carcinoma,
thyroid cancer, liver cancer, renal cancer, kaposis, sarcoma, and
another carcinoma or sarcoma. In an embodiment, the cancer is a
gynecological cancer, for example, a uterine cancer.
[0056] In an embodiment, the term "diagnosis," as used herein,
refers to any kind of procedure aiming to obtain information
instrumental in the assessment whether a patient suffers or is
likely or more likely than the average or a comparative subject,
the latter, for example, having similar symptoms, to suffer from
certain a disease or disorder in the past, at the time of the
diagnosis or in the future, to find out how the disease is
progressing or is likely to progress in the future or to evaluate
the responsiveness of a patient with regard to a certain treatment,
for example, the administration of immunosuppressive drugs. In
other words, the term "diagnosis" comprises not only diagnosing,
but also prognosticating and/or monitoring the course of a disease
or disorder.
[0057] In many cases the mere detection, in other words determining
whether or not detectable levels of the antibody are present in the
sample, is sufficient for the diagnosis. If the autoantibody can be
detected, this will be information instrumental for the clinician's
diagnosis and indicates an increased likelihood that the patient
suffers from a disease. In an embodiment, the relative
concentration of the antibody in the serum, compared to the level
that may be found in the average healthy subject, may be
determined. While in many cases it may be sufficient to determine
whether or not autoantibodies are present or detectable in the
sample, the method carried out to obtain information instrumental
for the diagnosis may involve determining whether the concentration
is at least 0.1, for example, 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50,
100, 200, 500, 1000, 10000 or 100000 times higher than the
concentration found in the average healthy subject.
[0058] The person skilled in the art will appreciate that a
clinician does usually not conclude whether or not the patient
suffers or is likely to suffer from a disease, condition or
disorders solely on the basis of a single diagnostic parameter, but
needs to take into account other aspects, for example, the presence
of other autoantibodies, markers, blood parameters, clinical
assessment of the patient's symptoms or the results of medical
imaging or other non-invasive methods such as polysomnography, to
arrive at a conclusive diagnosis. See Baenkler H. W. (2012),
General aspects of autoimmune diagnostics, in Renz, H., Autoimmune
diagnostics, 2012, de Gruyter, page 3. The value of a diagnostic
agent or method may also reside the possibility to rule out one
disease, thus allowing for the indirect diagnosis of another. In an
embodiment, the meaning of any symptoms or diseases referred to
throughout this application is in line with the person skilled in
the art's understanding as of Apr. 17, 2015 as evidenced by text
books and scientific publications.
[0059] Therefore, the term "diagnosis" does not, for example, imply
that the diagnostic methods or agents according to the present
disclosure will be definitive and sufficient to finalize the
diagnosis on the basis of a single test, let alone parameter, but
may refer to a contribution to what is referred to as a
"differential diagnosis," i.e., a systematic diagnostic procedure
considering the likelihood of a range of possible conditions on the
basis of a range of diagnostic parameters. Consequently, the
disclosed method, polypeptide or use, optionally for determining
whether a patient suffers from the a disease, may comprise
obtaining a sample from a patient, for example, a human patient,
determining whether an autoantibody binding to NCDN is present in
said sample, wherein said determining is performed by contacting
the sample with the disclosed polypeptide and detecting whether
binding occurs between said polypeptide and said autoantibody, for
example, using a labeled secondary antibody, for example, using a
method from the group comprising radioimmunoassay, Western blot,
line blot, ELISA, indirect and immunofluorescence, wherein said
autoantibody binds to said polypeptide if present in the sample,
and diagnosing the patient as suffering or being more likely to
suffer from said neurological disorder or cancer if the
autoantibody was determined to be present in the sample.
[0060] The term "diagnosis" may also refer to a method or agent
used to distinguish between two or more conditions associated with
similar or identical symptoms.
[0061] The term "diagnosis" may also refer to a method or agent
used to choose the most promising treatment regime for a patient.
In other words, the method or agent may relate to selecting a
treatment regimen for a subject. For example, the detection of
autoantibodies may indicate that an immunosuppressive therapy is to
be selected, which may include administrating to the patient one or
more immunosuppressive drugs.
[0062] The present disclosure relates to a complex comprising an
antibody, for example, autoantibody, binding to the disclosed
polypeptide. Such a complex may be used or detected as part of a
method for diagnosing a disease. A liquid sample comprising
antibodies from a subject may be used to practice the method. Such
a liquid sample may be any bodily fluid comprising a representative
set of antibodies from the subject, for example, a sample
comprising antibodies of the IgG immunglobulin class from the
subject. For example, a sample may be cerebrospinal fluid (CSF),
blood or blood serum, lymph, interstitial fluid and is, for
example, serum or CSF, for example, serum.
[0063] The step of contacting a liquid sample comprising antibodies
with the disclosed polypeptide may be carried out by incubating an
immobilized form of said polypeptide in the presence of the sample
comprising antibodies under conditions that are compatible with the
formation of the complex comprising said polypeptide and an
antibody, for example, an autoantibody, binding to the disclosed
polypeptide. The liquid sample, then depleted of antibodies binding
to the disclosed polypeptide may be removed subsequently, followed
by one or more washing steps. Finally the complex comprising the
antibody and the polypeptide may be detected. In an embodiment, the
term "conditions compatible with the formation of the complex" are
conditions that allow for the specific antigen-antibody
interactions to build up the complex comprising the polypeptide an
the antibody. In an embodiment, such conditions may comprise
incubating the polypeptide in sample diluted 1:100 in PBS buffer
for 30 minutes at 25.degree. C. In an embodiment, the term
"autoantibody," as used herein, refers to an antibody binding
specifically to an endogenous molecule of the animal, for example,
mammal, which produces said autoantibody, wherein the level of such
antibody is, for example, elevated compared the average of any
other antibodies binding specifically to such an endogenous
molecule. In an embodiment, the autoantibody is an autoantibody
binding to NCDN. Such an autoantibody may be isolated from samples
taken from patients suffering from the neurological disorder
characterized by two or more, for example, all symptoms selected
from the group comprising mixed movement disorder, acute decline of
visual performance, dysarthria and dysphagia.
[0064] In an embodiment, the detection of the complex for the
prognosis, diagnosis, methods or test kit according to the present
disclosure comprises the use of a method selected from the group
comprising immunodiffusion techniques, immunoelectrophoretic
techniques, light scattering immunoassays, light scattering
immunoassays, agglutination techniques, labeled immunoassays such
as those from the group comprising radiolabeled immunoassay, enzyme
immunoassays, chemiluminscence immunoassays, and immunofluorescence
techniques. The person skilled in the art is familiar with these
methods, which are also described in the state of the art, for
example, in Zane, H. D. (2001), Immunology--Theoretical &
Practical Concepts in Laboratory Medicine, W. B. Saunders Company,
in particular in Chapter 14.
[0065] Alternatively, a sample comprising tissue comprising the
disclosed polypeptide rather than a liquid sample may be used. The
tissue sample is, for example, from a tissue expressing endogenous
NCDN. Such a sample, which may be in the form of a tissue section
fixed on a carrier, for example, a glass slide for microscopic
analysis, may then be contacted with the disclosed antibody, for
example, autoantibody, binding to the disclosed polypeptide. The
antibody is, for example, labeled to allow for distinction from
endogenous antibodies binding to the disclosed polypeptide, so that
newly formed complexes may be detected and, optionally, quantified.
If the amount of complexes formed is lower than the amount found in
a sample taken from a healthy subject, the subject from whom the
sample examined has been taken is likely to suffer from a
disease.
[0066] Any data demonstrating the presence or absence of the
complex comprising the antibody and the disclosed polypeptide may
be correlated with reference data. For example, detection of said
complex indicates that the patient who provided the sample analyzed
has suffered, is suffering or is likely to suffer in the future
from a disease. If a patient has been previously diagnosed and the
method for obtaining diagnostically relevant information is run
again, the amount of complex detected in both runs may be
correlated to find out about the progression of the disease and/or
the success of a treatment. For example, if the amount of complex
is found to increase, this suggests that the disorder is
progressing, likely to manifest in the future and/or that any
treatment attempted is unsuccessful.
[0067] In an embodiment, a microplate, membrane ELISA, dot blot, or
line blot is used to carry out the diagnostic method according to
the disclosure. The person skilled in the art is familiar with the
experimental setup, which is described in the state of the art
(Raoult, D., and Dasch, G. A. (1989), The line blot: an immunoassay
for monoclonal and other antibodies. Its application to the
serotyping of gram-negative bacteria. J. Immunol. Methods, 125
(1-2), 57-65; WO2013041540). Briefly, the one or more antigen of
interest, in the case of the present disclosure the disclosed
polypeptide, may be attached to a carrier, for example,
nitrocellulose membrane, often in combination with additional
antigens and controls. The nitrocellulose carrier is subsequently
exposed to a sample comprising antibodies such as diluted serum. If
the sample comprises an antibody binding to the antigen, a complex
is formed which may be detected, for example, by incubation with a
secondary antibody binding to the constant region of the first
antibody, which secondary antibody comprises a detectable label,
for example, a radioactive isotope, a fluorescent dye or, in an
embodiment, an active enzyme fused or linked to the secondary
antibody, such as alkaline phosphatase, which may be readily
assayed using chromogenic substrates followed by simple visual
examination.
[0068] In another embodiment, the prognosis, diagnosis, methods or
test kit in line with the disclosed teachings contemplate the use
of indirect immunofluorescence. The person skilled in the art is
familiar with such techniques and the preparation of suitable
samples, which are described in the state of the art (U.S. Pat. No.
4,647,543; Voigt, J., Krause, C., Rohwader, E, Saschenbrecker, S.,
Hahn, M., Danckwardt, M., Feirer, C., Ens, K, Fechner, K, Barth, E,
Martinetz, T., and Stocker, W. (2012), "Automated Indirect
Immunofluorescence Evaluation of Antinuclear Autoantibodies on
HEp-2 Cells," Clinical and Developmental Immunology, vol. 2012,
doi:10.1155/2012/65105; Bonilla, E., Francis, L., Allam, F., et
al., Immuno-fluorescence microscopy is superior to fluorescent
beads for detection of antinuclear antibody reactivity in systemic
lupus erythematosus patients, Clinical Immunology, vol. 124, no. 1,
pp. 18-21, 2007). Briefly, a carrier, such as a cover glass for use
in microscopy, is coated with cells or tissue sections comprising
the antigen, in the case of the present disclosure the polypeptide
comprising one or more sequences of NCDN or a variant thereof. The
carrier comprising the antigen is exposed to a patient sample
comprising antibodies such as diluted serum. If the sample
comprises an antibody binding to the antigen, the resulting complex
may be detected, for example, by incubation with a secondary
antibody comprising a fluorescent dye such as fluorescein, followed
by visual examination using fluorescence microscopy. Suitable
reagents, devices and software packages are commercially available,
for example, from EUROIMMUN.TM., Lubeck, Germany.
[0069] A sample subjected to a test determining only whether an
autoantibody binding to NCDN is present, but in some embodiments,
the diagnostic methods, tests, devices and the like may contemplate
determining the presence of autoantibodies against a variety of
antigens relating to neurological autoimmune disease or variants
thereof, for example, selected from the group comprising Hu, Yo,
Ri, CV2, PNMA1, PNMA2, DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1,
CARPVIII, Zic4, Sox1, Ma, MAG, MP0, MBP, GAD65, amphiphysin,
recoverin, GABA A receptor, GABA B receptor, glycine receptor,
gephyrin, IgLON5, DPPX, aquaporin-4, MOG, NMDA receptor, AMPA
receptors, GRM1, GRM5, LGI1, VGCC and mGluR1 and CASPR2, which
antigens are for example, immobilized, for example, on a medical
device such as a line blot. The diagnostically relevant markers
ITPR1 (EP14003703.7), NBC1 (EP14003958.7) and ATP1A3, also referred
to as alpha 3 subunit of human neuronal Na(+)/K(+) ATPase
(EP14171561.5) have been described in the state of the art.
[0070] According to the teachings of the present disclosure, an
antibody, for example, an autoantibody binding to the disclosed
polypeptide used for the diagnosis of a disease is provided. The
person skilled in the art is familiar with methods for purifying
antibodies, for example, those described in Hermanson, G. T.,
Mallia, A. K., and Smith, P. K. (1992), Immobilized Affinity Ligand
Techniques, San Diego: Academic Press. Briefly, an antigen binding
specifically to the antibody of interest, which antigen is the
disclosed polypeptide, is immobilized and used to purify, via
affinity chromatography, the antibody of interest from an adequate
source. A liquid sample comprising antibodies from a patient
suffering from the neurological disorder identified by the
inventors may be used as the source.
[0071] According to the disclosure, an antibody, for example, an
autoantibody, is provided that is capable of binding specifically
to the disclosed polypeptide. In an embodiment, the term
"antibody," as used herein, refers to any immuglobulin-based
binding moieties, for example, one comprising at least one
immunoglobulin heavy chain and one immunoglobulin light chain,
including, but not limited to monoclonal and polyclonal antibodies
as well as variants of an antibody, in particular fragments, which
binding moieties are capable of binding to the respective antigen,
for example, binding specifically to it. In an embodiment, the term
"binding specifically," as used herein, means that the binding is
stronger than a binding reaction characterized by a dissociation
constant of 1.times.10-5 M, 1.times.10-7 M, 1.times.10-8 M,
1.times.10-9 M, 1.times.10-10 M, 1.times.10-11 M, or 1.times.10-12
M, as determined by surface plasmon resonance using BIACORE.TM.
equipment at 25.degree. C. in PBS buffer at pH 7. The antibody may
be isolated or in a mixture comprising further antibodies,
polypeptides, metabolites, cells and the like. In case the antibody
is an autoantibody, it may be part of an autoantibody preparation
which is heterogeneous or may be a homogenous autoantibody, wherein
a heterogeneous preparation comprises a plurality of different
autoantibody species as obtainable by preparation from the sera of
human donors, for example, by affinity chromatography using the
immobilized antigen to purify any autoantibody capable of binding
to said antigen. The antibody may be glycosylated or
non-glycosylated. The person skilled in the art is familiar with
methods that may be used for the identification, production and
purification of antibodies and variants thereof, for examples,
those described in EP 2 423 226 A2 and references therein. The
antibody may be used as a diagnostic agent, by itself, or in
combination, for example, in complex with the disclosed
polypeptide.
[0072] The present disclosure provides a method for isolating an
antibody, for example, an autoantibody, binding to the disclosed
polypeptide, comprising the steps a) contacting a sample comprising
the antibody with the disclosed polypeptide such that a complex is
formed, b) isolating the complex formed in step a), c) dissociating
the complex isolated in step b), and d) separating the antibody
from the disclosed polypeptide. A sample from a patient suffering
from the novel neurological disorder identified by the inventors
may be used as the source of antibody. Alternatively the antibody
may be a recombinant antibody. In some embodiments, the polypeptide
is immobilized, for example, on the matrix of a column suitable for
affinity chromatography or on a magnetic bead, since it is
straightforward to separate the complex comprising the polypeptide
and the antibody in step b) if such is the case. Subsequently, the
antibody may be separated from the immobilized antigen in step c),
for example, by eluting the antibody by addition of an excess of
non-immobilized antigen or by adding an agent interfering with
intramolecular interactions, for example, guanidinium chloride or
sodium chloride at a high concentration, the latter if that
electrostatic interactions are essential to maintain the complex.
One or more washing steps may be included to increase the purity of
the complex and the sensitivity and/or specificity of the assay
whenever the complex is formed as part of detection or purification
methods. The person skilled in the art is familiar with methods to
carry out each of these steps. Suitable methods are described in
the state of the art, for example, in the Handbooks "Affinity
chromatography," "Strategies for Protein Purification" and
"Antibody Purification" (2009/2010), published by GE Healthcare
Life Sciences, and in in Philips, Terry, M., Analytical techniques
in immunochemistry, 1992, Marcel Dekker, Inc.
[0073] The disclosure provides a pharmaceutical composition
comprising the disclosed polypeptide, which composition may be
suitable for administration to a subject, for example, a mammalian
subject, for example, to a human. Such a pharmaceutical composition
may comprise a pharmaceutically acceptable carrier. The
pharmaceutical composition may, for example, be administered
orally, parenterally, by inhalation spray, topically, by eyedrops,
rectally, nasally, buccally, vaginally or via an implanted
reservoir, wherein the term "parentally," as used herein, comprises
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intrasynovial, instrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques.
The pharmaceutical composition may be provided in suitable dosage
forms, for example, capsules, tablets and aqueous suspensions and
solutions, for example, in sterile form. It may be used in a method
of treatment of a disease, which method comprises administering an
effective amount of the disclosed polypeptide to a subject. In an
embodiment, the disclosure provides a vaccine comprising the
disclosed polypeptide, optionally comprising an auxiliary agent
such as an adjuvant or a buffer, and the use of the disclosed
polypeptide for the preparation of a vaccine.
[0074] Within the scope of the present disclosure, a medical or
diagnostic device comprising, for example, coated with the
disclosed (auto)antibody and/or the disclosed polypeptide is
provided. For example, such a medical or diagnostic device
comprises the disclosed polypeptide in a form that allows
contacting it with an aqueous solution, for example, the liquid
human sample, in a straightforward manner. In particular, the
disclosed polypeptide may be immobilized on the surface of a
carrier, which carrier comprises, but is not limited to glass
plates or slides, biochips, microtiter plates, beads, for example,
magnetic beads, chromatography columns, membranes or the like.
Exemplary medical devices include line blots, microplates and
biochips. In addition to the disclosed polypeptide, the medical or
diagnostic device may comprise additional polypeptides, for
example, positive or negative controls or known other antigens
binding to autoantibodies of diagnostic value, particularly those
related to other diseases associated with one or more identical or
similar symptoms.
[0075] The disclosed teachings provide a kit, for example, for
diagnosing a disease. Such a kit may comprise instructions
detailing how to use the kit and a means for contacting the
disclosed polypeptide with a bodily fluid sample from a subject,
for example, a human subject, for example, a line blot, wherein the
disclosed polypeptide is immobilized on the line blot. Furthermore,
the kit may comprise a positive control, for example, a batch of
autoantibody or recombinant antibody known to bind to the disclosed
polypeptide and a negative control, for example, a protein having
no detectable affinity to the disclosed polypeptide such as bovine
serum albumin. Finally, such a kit may comprise a standard solution
of the antibody or antigen for preparing a calibration curve.
[0076] In an embodiment, the kit comprises a means for detecting an
antibody, for example, an autoantibody, binding to the disclosed
polypeptide, for example, by detecting a complex comprising the
disclosed polypeptide and an antibody binding to the disclosed
polypeptide. Such means may be an agent that binds to said complex
and modifies the complex or carries a label such that makes the
complex detectable. For example, said means may be a labeled
antibody binding to said polypeptide, at a binding site other than
the binding site recognized by the primary antibody or to a
constant region of the primary antibody. Alternatively, said means
may be a secondary antibody binding to the constant region of the
autoantibody, for example, a secondary antibody specific for
mammalian IgG class antibodies. Alternatively, said means may be a
crosslinking reagent chemically linking the antibody and the
disclosed polypeptide, so the complex may be identified on account
of its increased molecular weight, for example, by SDS PAGE
followed by Coomassie staining or size-exclusion chromatography. A
multitude of methods and means for detecting such a complex have
been described in the state of the art, for example in Philips,
Terry, M., Analytical techniques in immunochemistry, 1992, Marcel
Dekker, Inc.
[0077] The disclosed polypeptide may be provided in the form of a
cell comprising and/or expressing a nucleic acid encoding said
polypeptide. If a nucleic acid comprising a sequence that encodes
for the disclosed polypeptide or variant thereof is used, such a
nucleic acid may be an unmodified nucleic acid. In an embodiment,
the nucleic acid is a nucleic acid that, as such, does not occur in
nature and comprises, compared to natural nucleic acid, at least
one modification, for example, an isotopic content or chemical
modifications, for example, a methylation, sequence modification,
label or the like indicative of synthetic origin. In an embodiment,
the nucleic acid is a recombinant nucleic acid or part or a nucleic
acid, and is, in an embodiment, part of a vector, in which it may
be functionally linked with a promoter that allows for expression,
for example, overexpression of the nucleic acid.
[0078] In an embodiment, said nucleic acid is inside a cell capable
of expressing it to the effect that the disclosed polypeptide or a
variant thereof is made and, for example, routed to the surface of
the cell. Said cell comprising the nucleic acid encoding the
disclosed polypeptide may be used according to the present
disclosure. The cell may be any kind of cell capable of expressing
the nucleic acid, for example, a prokaryotic or eukaryotic cell. In
an embodiment, the cell is a eukaryotic cell such as a yeast cell,
a eukaryotic cell from a multicellular organism, for example, an
insect cell, for example, a mammalian cell, for example, a mouse
cell, and, for example, a human cell.
[0079] The person skilled in the art is familiar with methods used
to synthesize, modify and amplify such a nucleic acid and to
transfect cells using such a nucleic acid, for example, in a vector
that allows for the transient or permanent maintenance or
expression of the nucleic acid in the cell. The person skilled in
the art is also familiar with a variety of suitable vectors, of
which are commercially available, for example, from ORIGENE.TM..
For example, a vector encoding for fusion constructs with a
C-terminal GFP may be used. The cell may be of eukaryotic or
prokaryotic origin and may be a mammalian cell, for example, a
HEK293, CHO or COS-7 cell. The cell comprising the nucleic acid
encoding for the disclosed polypeptide may be a recombinant cell or
an isolated cell wherein the term "isolated" means that the cell is
enriched such that, compared to the environment of the wild type of
said cell, fewer cells of other differentiation or species or in
fact no such other cells are present.
[0080] The disclosed teachings may not only be used for a
diagnosis, but also for preventing or treating a disease, more
specifically a method for preventing or treating a disease,
comprising the steps a) reducing the concentration of
autoantibodies binding to the disclosed polypeptide in the
subject's blood and/or b) administering one or more
immunosuppressive pharmaceutical substances, for example, selected
from the group comprising rituximab, prednisone,
methylprednisolone, cyclophosphamide, mycophenolatemofetil,
intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate,
azathioprine and/or the pharmaceutical composition.
[0081] It will be obvious to those having skill in the art that
many changes may be made to the details of the above-described
embodiments without departing from the underlying principles of the
disclosure. The scope of the present disclosure should, therefore,
be determined only by the following claims.
Sequence CWU 1
1
912190DNAHomo sapiensNeurochondrin coding sequence (Q9UBB6)
1atgtcgtgtt gtgacctggc tgcggcggga cagttgggca aggcgagcat catggcctcg
60gattgcgagc cagctctgaa ccaggcagag ggccgaaacc ccaccctgga gcgctacctg
120ggagccctcc gtgaggccaa gaatgacagc gagcagtttg cagccctgct
gctagtgacc 180aaggcagtca aagcaggtga catagatgcc aaaactcggc
ggcggatctt cgatgctgtc 240ggcttcacct tccccaatcg tctcctgacc
accaaggagg cgccggatgg ctgccctgac 300catgttctgc gggctttggg
tgtggccctg ctggcctgct tctgcagtga ccctgaactg 360gccgcccatc
cccaagtcct gaacaagatt cccattctta gcaccttcct cacagcccgg
420ggggacccgg acgatgctgc ccgccgctcc atgattgatg acacctacca
gtgcctgacg 480gctgtagcag gcacacccag agggcctcgg cacctcattg
ctggtggcac cgtgtctgcc 540ctatgccagg catacctggg gcacggctat
ggctttgacc aggccctggc actcctggtg 600gggctgctgg ctgctgccga
gacacagtgc tggaaggagg cggagcccga cctgctggcc 660gtgttgcggg
gcctcagtga ggatttccag aaagctgagg atgccagcaa gtttgagctc
720tgccagctgc tgcccctctt tttgcccccg acaaccgtgc cccctgaatg
ctaccgggat 780ctgcaggccg ggctggcacg catcctggga agcaagctga
gctcctggca gcgcaaccct 840gcactgaagc tggcagcccg cctggcacac
gcctgcggct ccgactggat cccggcgggc 900agctccggga gcaagttcct
ggccctgctg gtgaatctgg cgtgcgtgga agtgcggctg 960gcactggagg
agacgggcac ggaggtgaaa gaggatgtgg tgaccgcctg ctatgccctc
1020atggagttgg ggatccagga atgcactcgc tgtgagcagt cactgcttaa
ggagccacag 1080aaggtgcagc tcgtgagcgt catgaaggag gccatagggg
ctgttatcca ctacctgctg 1140caggtggggt cagagaagca gaaggagccc
tttgtgtttg cctcggtgcg gatcctgggt 1200gcctggctgg ccgaggagac
ctcatccttg cgtaaggagg tgtgccagct gctgcccttc 1260ctcgtccgct
atgccaagac cctctacgag gaggccgagg aggccaatga cctttcccag
1320caggtggcca acctggccat ctcccccacc accccagggc ccacctggcc
aggagacgct 1380ctccggctcc tcctgcctgg ctggtgccac ctgaccgttg
aagatgggcc ccgggagatc 1440ctgatcaagg aaggggcccc ctcgcttctg
tgcaagtatt tcctgcagca gtgggaactc 1500acatcccctg gccacgacac
ctcggtgctg cctgacagcg tggagattgg cctgcagacc 1560tgctgccaca
tcttcctcaa cctcgtggtc accgcaccgg ggctgatcaa gcgtgacgcc
1620tgcttcacat ctctaatgaa caccctcatg acgtcgctac cagcactagt
gcagcaacag 1680ggaaggctgc ttctggctgc taatgtggcc accctggggc
tcctcatggc ccggctcctt 1740agcacctctc cagctcttca gggaacacca
gcatcccgag ggttcttcgc agctgccatc 1800ctcttcctat cacagtccca
cgtggcgcgg gccaccccgg gctcagacca ggcagtgcta 1860gccctgtccc
ctgagtatga gggcatctgg gccgacctgc aggagctctg gttcctgggc
1920atgcaggcct tcaccggctg tgtgcctctg ctgccctggc tggcccccgc
tgccctgcgc 1980tcccgctggc cgcaggagct gctccagctg ctaggcagtg
tcagccccaa ctctgtcaag 2040cccgagatgg tggccgccta tcagggtgtc
ctggtggagc tggcgcgggc caaccggctg 2100tgccgggagg ccatgaggct
gcaggcgggc gaggagacgg ccagccacta ccgcatggct 2160gccttggagc
agtgcctgtc agagccctga 2190214PRTHomo sapiensNeurochondrin 377-390
2His Tyr Leu Leu Gln Val Gly Ser Glu Lys Gln Lys Glu Pro 1 5 10
350PRTHomo sapiensNeurochondrin 1-50 3Met Ser Cys Cys Asp Leu Ala
Ala Ala Gly Gln Leu Gly Lys Ala Ser 1 5 10 15 Ile Met Ala Ser Asp
Cys Glu Pro Ala Leu Asn Gln Ala Glu Gly Arg 20 25 30 Asn Pro Thr
Leu Glu Arg Tyr Leu Gly Ala Leu Arg Glu Ala Lys Asn 35 40 45 Asp
Ser 50 419PRTHomo sapiensNeurochondrin 711-729 4Glu Glu Thr Ala Ser
His Tyr Arg Met Ala Ala Leu Glu Gln Cys Leu 1 5 10 15 Ser Glu Pro
5150PRTHomo sapiensNeurochondrin 1-150 5Met Ser Cys Cys Asp Leu Ala
Ala Ala Gly Gln Leu Gly Lys Ala Ser 1 5 10 15 Ile Met Ala Ser Asp
Cys Glu Pro Ala Leu Asn Gln Ala Glu Gly Arg 20 25 30 Asn Pro Thr
Leu Glu Arg Tyr Leu Gly Ala Leu Arg Glu Ala Lys Asn 35 40 45 Asp
Ser Glu Gln Phe Ala Ala Leu Leu Leu Val Thr Lys Ala Val Lys 50 55
60 Ala Gly Asp Ile Asp Ala Lys Thr Arg Arg Arg Ile Phe Asp Ala Val
65 70 75 80 Gly Phe Thr Phe Pro Asn Arg Leu Leu Thr Thr Lys Glu Ala
Pro Asp 85 90 95 Gly Cys Pro Asp His Val Leu Arg Ala Leu Gly Val
Ala Leu Leu Ala 100 105 110 Cys Phe Cys Ser Asp Pro Glu Leu Ala Ala
His Pro Gln Val Leu Asn 115 120 125 Lys Ile Pro Ile Leu Ser Thr Phe
Leu Thr Ala Arg Gly Asp Pro Asp 130 135 140 Asp Ala Ala Arg Arg Ser
145 150 6150PRTHomo sapiensNeurochondrin 151-300 6Met Ile Asp Asp
Thr Tyr Gln Cys Leu Thr Ala Val Ala Gly Thr Pro 1 5 10 15 Arg Gly
Pro Arg His Leu Ile Ala Gly Gly Thr Val Ser Ala Leu Cys 20 25 30
Gln Ala Tyr Leu Gly His Gly Tyr Gly Phe Asp Gln Ala Leu Ala Leu 35
40 45 Leu Val Gly Leu Leu Ala Ala Ala Glu Thr Gln Cys Trp Lys Glu
Ala 50 55 60 Glu Pro Asp Leu Leu Ala Val Leu Arg Gly Leu Ser Glu
Asp Phe Gln 65 70 75 80 Lys Ala Glu Asp Ala Ser Lys Phe Glu Leu Cys
Gln Leu Leu Pro Leu 85 90 95 Phe Leu Pro Pro Thr Thr Val Pro Pro
Glu Cys Tyr Arg Asp Leu Gln 100 105 110 Ala Gly Leu Ala Arg Ile Leu
Gly Ser Lys Leu Ser Ser Trp Gln Arg 115 120 125 Asn Pro Ala Leu Lys
Leu Ala Ala Arg Leu Ala His Ala Cys Gly Ser 130 135 140 Asp Trp Ile
Pro Ala Gly 145 150 7150PRTHomo sapiensNeurochondrin 301-450 7Ser
Ser Gly Ser Lys Phe Leu Ala Leu Leu Val Asn Leu Ala Cys Val 1 5 10
15 Glu Val Arg Leu Ala Leu Glu Glu Thr Gly Thr Glu Val Lys Glu Asp
20 25 30 Val Val Thr Ala Cys Tyr Ala Leu Met Glu Leu Gly Ile Gln
Glu Cys 35 40 45 Thr Arg Cys Glu Gln Ser Leu Leu Lys Glu Pro Gln
Lys Val Gln Leu 50 55 60 Val Ser Val Met Lys Glu Ala Ile Gly Ala
Val Ile His Tyr Leu Leu 65 70 75 80 Gln Val Gly Ser Glu Lys Gln Lys
Glu Pro Phe Val Phe Ala Ser Val 85 90 95 Arg Ile Leu Gly Ala Trp
Leu Ala Glu Glu Thr Ser Ser Leu Arg Lys 100 105 110 Glu Val Cys Gln
Leu Leu Pro Phe Leu Val Arg Tyr Ala Lys Thr Leu 115 120 125 Tyr Glu
Glu Ala Glu Glu Ala Asn Asp Leu Ser Gln Gln Val Ala Asn 130 135 140
Leu Ala Ile Ser Pro Thr 145 150 8150PRTHomo sapiensNeurochondrin
451-600 8Thr Pro Gly Pro Thr Trp Pro Gly Asp Ala Leu Arg Leu Leu
Leu Pro 1 5 10 15 Gly Trp Cys His Leu Thr Val Glu Asp Gly Pro Arg
Glu Ile Leu Ile 20 25 30 Lys Glu Gly Ala Pro Ser Leu Leu Cys Lys
Tyr Phe Leu Gln Gln Trp 35 40 45 Glu Leu Thr Ser Pro Gly His Asp
Thr Ser Val Leu Pro Asp Ser Val 50 55 60 Glu Ile Gly Leu Gln Thr
Cys Cys His Ile Phe Leu Asn Leu Val Val 65 70 75 80 Thr Ala Pro Gly
Leu Ile Lys Arg Asp Ala Cys Phe Thr Ser Leu Met 85 90 95 Asn Thr
Leu Met Thr Ser Leu Pro Ala Leu Val Gln Gln Gln Gly Arg 100 105 110
Leu Leu Leu Ala Ala Asn Val Ala Thr Leu Gly Leu Leu Met Ala Arg 115
120 125 Leu Leu Ser Thr Ser Pro Ala Leu Gln Gly Thr Pro Ala Ser Arg
Gly 130 135 140 Phe Phe Ala Ala Ala Ile 145 150 9129PRTHomo
sapiensNeurochondrin 601-729 9Leu Phe Leu Ser Gln Ser His Val Ala
Arg Ala Thr Pro Gly Ser Asp 1 5 10 15 Gln Ala Val Leu Ala Leu Ser
Pro Glu Tyr Glu Gly Ile Trp Ala Asp 20 25 30 Leu Gln Glu Leu Trp
Phe Leu Gly Met Gln Ala Phe Thr Gly Cys Val 35 40 45 Pro Leu Leu
Pro Trp Leu Ala Pro Ala Ala Leu Arg Ser Arg Trp Pro 50 55 60 Gln
Glu Leu Leu Gln Leu Leu Gly Ser Val Ser Pro Asn Ser Val Lys 65 70
75 80 Pro Glu Met Val Ala Ala Tyr Gln Gly Val Leu Val Glu Leu Ala
Arg 85 90 95 Ala Asn Arg Leu Cys Arg Glu Ala Met Arg Leu Gln Ala
Gly Glu Glu 100 105 110 Thr Ala Ser His Tyr Arg Met Ala Ala Leu Glu
Gln Cys Leu Ser Glu 115 120 125 Pro
* * * * *