WO2008085035A1 - Peptide markers for diagnosis of neurodegenerative disease - Google Patents

Abstract

The present invention relates to a method for detecting neurodegenerative disease for example multiple sclerosis or CIS, comprising determining. the expression level of chromogranin A in blood, cerebrospinal fluid or nervous tissue. The invention further relates to a marker for detecting neurodegenerative disease wherein said marker is chromogranin A.

Description

Title: Peptide markers for diagnosis of neurodegenerative disease

FIELD OF THE INVENTION

The present invention is in the field of disease diagnostics. In particular, the invention relates to the detection of peptides and/or proteins as markers for the diagnosis, prognosis, or therapeutic monitoring of neurodegenerative disease such as multiple sclerosis and clinically isolated syndrome of demyelination. The invention further provides the use of chromogranin A as a marker for the diagnosis, prognosis, or (therapeutic) monitoring of neurodegenerative disease.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MScI) is a complex dise ase of the central nervous system (CNS) with presumed autoimmune origin. Its pathology is characterized by a combination of inflammation, demyelination and axonal damage. These processes are not uniformly represented across patient populations but can predominate selectively in individual patients, contributing to the heterogeneity of phenotypic expression of the disease.

Due to the complex nature of the disease, there is probably no single protein or peptide that can serve as a biomarker for MScI in a clinically relevant way. However, individuals with MScI might be differentiated from healthy individuals by a proteomic pattern consisting of a set of distinct individual proteins or peptides that are not independently reliable indicators of disease state.

Because the disease process in MScI is located in the CNS, cerebrospinal fluid (CSF) is a promising body fluid in which to search for biomarkers and dise ase -associated proteins and peptides. The absence of an active clotting system in CSF compared to serum makes it an attractive bio- fluid to perform proteomic studies. Biomarkers for MScI could potentially identify relevant biological pathways for MScI, which could help clarify currently unsolved issues in the pathology and etiology of the disease. Additionally, biomarkers could also be useful as a new method for diagnosis of MScI, which might enable earlier detection of the disease, or as a tool to identify clinically relevant subgroups. Earlier detection, and subsequently early treatment, will retard the long-term evolution of the disease.

SUMMARY OF THE INVENTION

By using a combination of different MALDI-MS techniques for marker detection, the present inventors have now found that chromogranin A is a powerful biomarker for neurodegenerative disease in general and for MScI and CIS in particular.

In a first aspect, the present invention provides a method for detecting neurodegenerative disease in a subject, comprising determining the expression level of the protein chromogranin A in blood, cerebrospinal fluid or nervous tissue.

In a preferred embodiment of said method, said expression level is determined in a sample of blood, cerebrospinal fluid or nervous tissue of said subject, most preferably cerebrospinal fluid. In another preferred embodiment of said method, said neurodegenerative disease is multiple sclerosis or clinically isolated syndrome of demyelination (CIS).

The present invention also provides a method as described above, wherein in addition to said expression level of chromogranin A, also the expression level of one or more of the proteins selected from, clusterin, complement C3, complement C4B, beta V spectrin, hypothetical protein XP_011125 (osteopontin), apolipoprotein D, complement C4A, contactin 1, neuronal pentraxin receptor and RNA binding motif protein is determined.

In a preferred embodiment of said method, said expression level is determined by detecting the chromogranin A protein or the additional protein, or a peptide fragment thereof in a mass range of 800 to 27,000 Da. Also, a transcription product of the chromogranin A gene or from the additional proteins described above, such as an mRNA from the chromogranin A gene or one or more of the additional proteins, may be determined in order to determine the required expression level.

In methods of the present invention, the peptides are suitably detected by MALDI MS analysis and therefore will generally be digested, for instance by trypsin, and detected when having a molecular mass in a range of 400-20,000, preferably in a range of 800 to 4,000 Da. The nucleic acids, such as the mRNAs transcribed from the chromogranin A gene may be detected for instance with RT-PCR (reverse-transcriptase polymerase chain reaction) optionally in combination with a suitable method for detecting DNA amplification products produced in such a reaction.

In a preferred embodiment of said method, said detection is performed by immunoassay or mass spectrometry.

In another aspect, the present invention provides the use of a method for detecting neurodegenerative disease as described above, wherein said use is for monitoring a disease process or a response to a disease therapy. Therapeutic monitoring of disease means monitoring of disease activity and treatment response.

In a preferred embodiment of said use, said disease is multiple sclerosis or CIS.

In another aspect, the present invention provides a marker protein or marker peptide for detecting neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject wherein said marker protein is the expression level of the protein chromogranin A in blood, cerebrospinal fluid or nervous tissue and wherein said marker peptide is a peptide fragment of chromogranin A having a mass of between 800 and 27,000 Da.

In yet another aspect, the present invention provides a marker profile for detecting neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject wherein said marker profile comprises the expression level in blood, cerebrospinal fluid or nervous tissue of a subject of a first protein being chromogranin A or a peptide thereof, and wherein said marker profile further comprises at least one additional expression level of a protein or peptide fragment selected from the group of clusterin, complement C3, complement C4B, beta V spectrin, osteopontin, apolipoprotein D, complement C4A, contactin 1, neuronal pentraxin receptor and RNA binding motif protein.

A marker profile is thus the combined expression profile, determined as amount of protein or as amount of gene transcript, of a number of markers specific for neurodegenerative disease, in particular multiple sclerosis and/or CIS, as disclosed herein.

In yet another aspect, the present invention provides for the use of a marker or marker profile of the invention for the detection of neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject. It is known in the art of proteome analysis that factors such as sample stability and a low number of measurements per sample can cause difficulties regarding the reproducibility of proteomic profiling studies. Also, it is known that there is low reproducibility of peak height in MALDI-TOF MS. The method of the present invention overcomes these problems in several ways and is less affected by these variations. First, the samples are all handled in a standardized way. Secondly, the sample preparation method is uncomplicated and straightforward. Thirdly, the height of the peaks is not included in the analysis because quantitative measurements of peak heights with MALDI TOF MS are poorly reproducible, with standard deviations up to 30%. In the present method only the absence or presence of the peaks is scored, (see the Examples below for details)

Preferred embodiments of the method of the present invention include for instance the detection of the marker protein or marker peptide in a sample of nervous tissue, CSF or blood (or serum) of a subject by MALDI-FT mass spectrometry, MALDI Triple-quad mass spectrometry or an immunoassay, such as ELISA or immunohistochemistry. The tissue or fluid sample is prepared for such analyses by methods well known to the skilled person.

Samples used in aspects of the present invention may be obtained by biopsy or puncture, involving the removal of a small portion of tissue from the body, such as needle biopsy or open biopsy. Alternatively, the sample may be body liquids such as blood, serum, liquor, cerebrospinal fluid or the like.

Samples used in aspects of the present invention may be unprocessed, or processed samples, meaning that the samples may or may not have been subjected to procedures wherein the biological, physical or chemical composition of the sample is altered. The samples may also be subjected to multiple processing steps.

In an alternative embodiment of a method of the invention, the optionally processed samples are body tissue samples processed by subjecting said samples to laser capture microdissection to provide collections of microdissected cells, said collections preferably amounting to about 200- 3,000 cells. Preferably, said collections of microdissected cells are provided in the form of pooled collections of microdissecte d cells.

In yet another alternative embodiment of a method of the invention the optionally processed samples are body tissue samples, body fluid samples, or collections of microdissected cells, optinally processed by subjection to protein digestion, preferably using trypsin, to provide optionally processed samples comprising proteins or peptide fragments from the proteins in said samples. Thus, the method optionally comprises the step of cleaving the proteins in a sample (i.e. polypeptides in general) with a (optionally sequence specific) cleavage agent to form peptide fragments, optionally followed by deactivating the cleavage agent. A sequence specific cleavage agent in aspects of the present invention preferably cleaves the polypeptides on the C-terminal side of a lysine residue. The specific cleavage agent preferably comprises Lys-C or trypsin. The cleavage agent is preferably trypsin. Polypeptide cleaving (e.g. trypsin digestion) is performed to provide peptide fragments sufficiently small to be analysed by MALDI analysis. However, some samples may comprise peptide fragments of sufficiently small size to allow direct MALDI analysis. Examples of peptides that can be detected or analyses in unprocessed samples include (neuro)peptides, hormones, etc.

In principle, any body tissue of a subject may be used in aspects of the invention. Suitably a body tissue is selected from the group consisting tissues of brain, lung, heart, prostate, esophagus, stomach, jejunum, ileum, caecum, colon, gall bladder, bile duct, breast, ovary, testicle, lymph node, thymus, kidney, liver, muscle, nerve, bone, bone marrow, and placenta. A highly preferred tissue sample is a nerve tissue sample.

The body fluid analysed in a method of the present invention may suitably be selected from the group consisting of blood, serum, cerebrospinal fluid (CSF), urine, saliva and semen. Highly preferred fluid samples are blood, serum, and cerebrospinal fluid (CSF).

Body fluid samples, when used in methods of the invention, may suitably be provided in sample volumes of between 0.01 and 100 μl. However, it is a particular advantage of the present invention that very small sample volumes will generally suffice. An amount in a range from 0.1-25 μl, preferably in a range from 1-10 μl of optionally processed body fluid is generally sufficient for MALDI- FT-ICR mass spectrometric analysis. A suitable sample fluid preferably comprises about 0.05- 5 mg/ml of protein.

Herein below, the terms "patient" and "subject" are used interchangeably to indicate animal subjects, including human and non-human subjects that are in need of disease diagnosis.

In the various methods described in the present invention the step of detecting the marker peptide or marker protein in a sample may suitably be performed by MALDI Triple-quad analysis of proteins and peptides in a sample to quantify said marker protein or marker peptide indicative for a specific disease in a subject. SHORT DESCRIPTION OF THE DRAWINGS

Figures 1-6 show p-value bar charts of all comparisons Graphical representation of the results of the Wilcoxon-Mann- Whitney tests for the comparisons of the four groups. The heights of the bars represent the total number of peaks with a specific p-value interval, whereas the green bar indicates the number of peaks that were predominantly present in the first mentioned group, and the blue bar indicates the number of peaks predominantly present in the second mentioned group. The number of peaks found for each p-value after randomization is represented by the red line, which can be viewed as a baseline.

Any low p-value bar that exceeds the height of the baseline indicates statistically significant differences in that particular comparison. Figure 1 shows the comparison of MScI with other neurological disease (OND), in which the large skewing to the left of the p value range indicates that these groups differ significantly. The same pattern can be seen in figure 2, which represents the comparison of CIS with OND. Smaller, but still significant differences can be observed in the comparison of MScI with other inflammatory neurological disease (OIND) and the comparison of CIS with OIND, in figures 3 and 4, respectively. The comparison of MScI with CIS (figure 5) and the comparison of OIND with OND (figure 6) do not show significant differences.

Figure 7 shows the amino acid sequence of human chromogranin A. Figure 8 shows the amino acid sequence of human clusterin. Figure 9 shows the amino acid sequence of human complement component 3, isoform CRA_a.

Figure 10 shows the amino acid sequence of complement component 4B (also known as: Complement C4-B precursor (Basic complement C4) [Contains: Complement C4 beta chain; Complement C4-B alpha chain; C4a anaphylatoxin; C4b-B; C4d-B; Complement C4 gamma chain]). Figure 11 shows the amino acid sequence of human beta V spectrin (also known as: human spectrin, beta, non-erythrocytic 5).

Figure 12 shows the amino acid sequence of human osteopontin (also known as: hypothetical protein XP_011125 and human secreted phosphoprotein 1 isoform a).

Figure 13 shows the amino acid sequence of human apolipoprotein D.

Figure 14 shows the amino acid sequence of human complement C4A (also known as: Complement component 4A (Rodgers blood group). Figure 15 shows the amino acid sequence of human contactin 1.

Figure 16 shows the amino acid sequence of human neuronal pentraxin receptor.

Figure 17 shows the amino acid sequence of human RNA binding motif protein 7. Figure 18 shows the amino acid sequence of Ig kappa chain V-III region SIE (UniProtKB/Swiss-Prot entry P01620).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "chromogranin A (CgA)" as used herein, refers to parathyroid secretory protein 1 (gene name CHGA). The protein is a member of the chromogranin/secretogranin (granins) family of neuroendocrine secretory proteins, i.e. it is located in secretory vesicles of neurons and endocrine cells, and is the precursor to several functional peptides including vasostatin, pancreastatin, catestatin and parastatin. The term "chromogranin A", as used herein, refers in particular to the protein essentially having the amino acid sequence as shown in Fig. 7. The term includes reference to other isoforms. The skilled person will understand that deviations and mutation may occur within the amino acid sequence or gene sequence of chromogranin A, which deviations and mutations are encompassed in the term chromogranin A as used herein. Also, the skilled person will understand from this specification that peptide fragments of the chromogranin A protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of chromogranin A having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "clusterin" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 8. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the clusterin protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of clusterin having a length of 7-100 amino acids, preferably 10-30 amino acids. The term "complement component 3" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 9. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the complement component 3 protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of complement component 3 having a length of 7-100 amino acids, preferably 10- 30 amino acids.

The term "complement component 4B" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 10. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the complement component 4B protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of complement component 4B having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "beta V spectrin" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 11. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the beta V spectrin protein can also be detected and serve as markers, such- as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of beta V spectrin having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "osteopontin" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 12. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the osteopontin protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of osteopontin having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "apolipoprotein D" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 13. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the apolipoprotein D protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of apolipoprotein D having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "complement component 4A" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 14. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the complement component 4A protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of complement component 4A having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "contactin 1" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 15. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the contactin 1 protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of contactin lhaving a length of 7-100 amino acids, preferably 10-30 amino acids. The term "neuronal pentraxin receptor" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 16. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the neuronal pentraxin receptor protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of neuronal pentraxin receptor having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "RNA binding motif protein 7" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 17. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the RNA binding motif protein 7 protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of RNA binding motif protein 7 having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "Ig kappa chain V-III region SIE" as used herein refers to the protein essentially having the amino acid sequence as shown in Fig. 18. The term includes reference to other isoforms. When reference is made to the marker, peptide fragments of the Ig kappa chain V-III region SIE protein can also be detected and serve as markers, such as peptides of the protein obtained by enzymatic (tryptic) digestion of samples of a subject wherein said marker is to be detected. In particular, suitable peptide fragments are fragments of Ig kappa chain V-III region SIE having a length of 7-100 amino acids, preferably 10-30 amino acids.

The term "neurodegenerative disease" includes all diseases accompanied by degeneration of nerve cells, and is not limited by its cause. The neurodegenerative disease in the present invention also includes neuropathy or disease in need of nerve regeneration. The nerve cell may be any type of nerve cells in the living body, including, for example, central nerves (e.g., cerebral nerves, spinal nerves, etc.), peripheral nerves (e.g., autonomic nervous system (e.g., sympathetic nerve, parasympathetic nerve, etc.), etc.) and so on. The neurodegenerative disease is, for example, a disease of central nerve, including Parkinson's disease, Parkinson syndrome,

Alzheimer's disease, Down's disease, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, olivopontocerebellar atrophy, cortical basal degeneration, familial dementia, frontal temporal dementia, senile dementia, diffuse Lewy body disease, striatonigral degeneration, chorea athetosis, dystonia, Meigs's syndrome, late cortical cerebellar atrophy, familial spastic paraplegia, motor neuron disease, Machado-Joseph disease, Pick's disease, nervous dysfunction after cerebral apoplexy (for example, brain hemorrhage (e.g., hypertensive intracerebral bleeding, etc.), cerebral infarction (e.g., cerebral thrombosis, cerebral embolus, etc.), transient ischemic attack, subarachnoid hemorrhage, etc.), nervous dysfunction after spinal damage, demyelinating disease (for example, multiple sclerosis, Guillain-Barre syndrome, acute disseminated encephalomyelitis, acute cerebellitis, transverse myelitis, etc.), brain tumor (for example, astrocytoma, etc.), cerebrospinal disease caused by infection (for example, meningitis, cerebral abscess, Creutzfeldt-Jakob disease, AIDS dementia, etc.), mental disease (schizophrenia, manic-depressive psychosis, neurosis, psychosomatic disease, epilepsy, etc.) and the like. The neurodegenerative disease is more preferably selected from Parkinson's disease, Parkinson syndrome, Alzheimer's disease, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, and demyelinating disease, still more preferably, from multiple sclerosis, Guillain-Barre syndrome, acute disseminated encephalomyelitis, acute cerebellitis, transverse myelitis, and clinically isolated syndrome of demyelination. The term "nervous tissue" as used herein refers to tissue made up of neurons and includes tissue of the brain, spinal cord and peripheral nervous system.

The term "determining the expression level of the protein" as used herein refers to measuring absolute or relative amounts of protein, and to absolute or relative amounts of transcription products transcribed from the genes encoding said protein.

Markers for detecting neurodegenerative disease in a patient

The present inventors set out to examine CSF peptide profiles of MScI patients in order to identify the peptides and proteins that differ between the patient groups. Using MALDI-TOF Mass Spectrometry they analyzed a total of 164 CSF samples. After tryptic digestion of the CSF proteins, the peptide mixtures were measured by MALDI-TOF MS and subsequently analyzed using a bioinformatics tool. MALDI Mass spectrometry was used because it is well suited for high-throughput peptide profiling, as it is a relatively fast and accurate way of analyzing complex protein and peptide mixtures. Although it is also a useful tool for the identification of peptides and proteins, the inventors also confirmed the identifications by more advanced mass spectrometry techniques (MALDI-FTMS and ESI-Orbitrap). The ESI- orbitrap also identified an additional eight peptides that were not identified by the MALDI-TOF/TOF experiments.

The present inventors identified several proteins that were specifically expressed in multiple sclerosis and CIS relative to other inflammatory neurological disease or some other neurological disease (see Example 1) by using a method comprising the following steps:

(a) providing an optionally processed (e.g. trypsin digested) sample of a diseased body tissue or fluid as a test sample (i.e. a glioma), and an optionally processed sample of a corresponding healthy body tissue or fluid as a reference sample, wherein said samples comprise peptides and/ or proteins; (b) subjecting both test and reference sample to MALDI- FT-ICR mass spectrometry to generate mass spectra for individual peptides in each sample and to quantify the amount of individual peptides present in each sample;

(c) comparing the amount of an individual peptide present in the test sample with the amount of a peptide having a corresponding mass in the reference sample to generate a list of peptides differentially expressed between test and reference sample, and

(d) subjecting the test and/or reference sample of step (a) to tandem mass spectrometry (MS-MS), in order to identify the differentially expressed peptides and/or the proteins from which they derive thus providing a candidate marker protein or marker peptide.

In this method, microdissected hypertrophied and normal blood vessels of the brain were used. The peptides of the enzymatically digested proteins derived from the small numbers of cells obtained by microdissection, were measured by MALDI-FT mass spectrometry. The identification of differentially expresses peptides was achieved by combining nano-LG fractionation of samples with offline MALDI-TOF/TOF and MALDI FTMS measurements. The findings were validated by using specific antibodies. Details of these experiments are described in the Examples below. By using the above method, the inventors discovered a proteinaceous marker, chromogranin A, the expression level of which was indicative for multiple sclerosis and CIS.

In addition to chromogranin A, also identified as markers indicative for multiple sclerosis and CIS were clusterin, complement C3, complement C4B, beta V spectrin, osteopontin, apolipoprotein D, complement C4A, contactin 1, neuronal pentraxin receptor, RNA binding motif protein and Ig kappa chain V-III region SIE, and these markers may be used instead of chromogranin A in exactly the same manner in all aspects of the invention as disclosed herein. A total of 164 CSF samples taken from neurological patients were classed into four groups according to the clinical diagnosis: multiple sclerosis (MScI, n=44), clinically isolated syndrome of demyelination (CIS, n=40), other inflammatory neurological disease (OIND, n=26) and other neurological disease (OND, n=54). After tryptic digestion, the samples were measured by MALDI-TOF

MS. Spectra were analysed using the R-project software package, in which a peak detection algorithm was developed. Subsequently the peak lists were compared based on ranked data (non-parametric). Significant differences were observed in the comparisons of MScI vs. OND and CIS vs. OND. The comparisons of MScI vs. OIND, and CIS vs. OIND showed fewer significant differences. No significant differences were found in comparisons MScI vs. CIS and OIND vs. OND. MScI and CIS had strikingly similar profiles, probably a reflection of common pathological mechanisms. Three differentially expressed proteins in the comparison of MScI vs. OND were identified: chromogranin A, a potential marker for neurodegeneration; and two important factors in complement mediated inflammatory reaction, clusterin and complement C3. CSF chromogranin A levels were confirmed to be significantly elevated in the MScI group using an ELISA.

Suitable body fluid samples wherein the expression level of a marker of the invention may be detected include blood, serum or, most preferably, cerebrospinal fluid samples. The body tissue sample wherein the expression level of the markers may be detected may be any body tissue, preferably however, the tissue is a nervous tissue.

The marker of the present invention is very suitably used in a method for monitoring the disease activity of neurodegenerative disease or the response of the patient to treatment regimens aimed at ameliorating or preventing neurodegenerative disease. Such a method comprises the step of measuring the expression level of chromogranin A in a body fluid or tissue. Reference values for markers may be determined as described below and methods of diagnosis of neurodegenerative disease may be performed as described in the Examples below.

THe markers of the present invention may also be used to differentiate between various neurological disorders. It was found that, on the basis of proteomic CSF analysis, the group of MScI patients could be clearly distinguished from the noninflammatory neurological controls (patients with isolated headaches; neurological degeneration; non neurological disease; infarct; brain tumor). Since MScI is considered to be an inflammatory disease, a group of controls covering other CNS inflammatory neurological diseases was included. The inventors found that it was also possible to clearly differentiate between the CSF proteomic profiles of the MScI patients and the inflammatory neurological controls (infections [(bacterial and sterile], vasculitis and Guillain Barre Syndrome). As MScI has both an inflammatory and a neurodegenerative component, comparison with both control groups was considered important to ensure that the control groups covered both the inflammatory and the non- inflammatory phase of the disease. It was found that it was also possible to make similar distinctions in the proteomic CSF analyses of the CIS patients when compared to the non-inflammatory controls, as well as in the comparison of the CIS patients with the inflammatory neurological controls. The markers which may be used for this purpose are provide in Tables 2 and 3 in the Example below.

Generally, the marker chromogranin A is detected in amounts of around 100 to 300 U/L when using the ELISA method as described in the Example. The skilled person will understand that the exact value will depend on the marker detected, on the sample type, and on the reference values measured in samples obtained from normal, healthy subjects. The skilled artisan is well aware of methods to obtain reference values for diagnostic markers. Generally, typical reference samples will be obtained from subjects that are clinically well documented and that are free from the disease. In such samples, normal (reference) concentrations of the marker proteins can be determined, for instance by providing the average concentration over the reference population. In determining the reference concentration of the marker a variety of considerations is taken into regard. Among such considerations are the type of disease to be diagnosed, the location of disease and the type of sample involved (e. g., tissue or CSF), the patient's age, weight, sex, general physical condition and the like. For instance, a group of at least 2 to preferably more than 3 subjects, preferably ranked according to the above considerations, for instance from various age categories, are taken as reference group.

The marker of the present invention is absent in samples wherein no neurodegenerative disease is present. In contrast, the marker is present in samples wherein neurodegenerative disease is clinically manifest. For instance in the case of multiple sclerosis, the chromogranin A protein can easily be detected at elevated levels in the CSF by immunological techniques, whereas in CSF of healthy subjects, said marker is present at markedly lower concentrations (See Table 4 in the Example). In general, a level in the concentration of the marker that is increased at least 1.1-10 times, preferably 1.5-5 times, but suitably about 2 times, relative to concentration of the reference value is indicative of the presence of neurodegenerative disease. Depending on the normal (healthy) status, a marker indicative of neurodegenerative disease as defined herein may be present in the diseased condition vs. absent in the normal condition. More often however, the level of expression of the marker will be altered, usually enhanced, so that elevated levels of the marker indicate the presence of the disease or even the severity of the disease condition. Therefore, in some instances, quantitative detection of the chromogranin A marker and comparison with reference values is necessary in order to draw conclusions. The steps which must be taken in order for a diagnosis to be made are generally: i) an examination phase involving the collection of data, ii) a comparison of these data with standard values, iii) a finding of any significant deviation during the comparison, and iv) the attribution of the deviation to a particular clinical picture, i.e. the deductive medical or veterinary decision phase.

In methods of the present invention, step iv is generally excluded. The methods of the present invention in particular relate to the technical steps of providing samples and providing clinical data on marker concentrations, which steps proceed the deductive medical or veterinary decision phase.

Detection of the marker in a patient sample may be performed by any method available to the artisan. Generally, in order to detect the subtle concentration differences in the expression level of the marker, sophisticated methods are required. The skilled person is well acquainted with the various methods available, and these need not be described in great detail here.

In short, suitable methods include mass spectrometric methods such as those described and used herein, in particular in the Examples, and immunological detection methods. Immunological detection methods (i.e. immunoassays) for determining the (quantitative) presence of a peptide or protein in a sample are well known to those of skill in the art. The markers identified by methods of the present invention can be employed as immunogens for constructing antibodies immunore active to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.

In another aspect, the present invention provides for the use of a disease marker, identified by a method for identifying a disease marker according to the invention, in diagnosis, prognosis, or therapeutic monitoring of neurodegenerative disease.

Polyclonal and monoclonal antibodies raised against chromogranin A protein or peptide fragments thereof and that bind specifically thereto can be used for detection purpose in the present invention, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. A variety of immunoassay formats may be used to select antibodies specifically reactive with a particular peptide or protein marker. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine selective binding. Examples of types of immunoassays that can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.

Detection of the peptide or protein marker using an antibody can be done utilizing immunoassays that are run in either the forward, reverse, or simultaneous modes, including immunological assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

Immunological detection may for instance be performed by ELISA using commercial tests for detection of chromogranin A in for instance CSF samples (DakoCytomation, Denmark).

Antibodies can be bound to many different carriers and used to detect the presence of the disease markers. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.

The binding of the antibody to the marker of the present invention can be detected in numerous ways that are well known in the art. Binding of the antibody and disease marker forms an immune complex that can be detected directly or indirectly. The immune complexes are detected directly, for example, when the antibodies employed are conjugated to a label. The immune complex is detected indirectly by examining for the effect of immune complex formation in an assay medium on a signal producing system or by employing a labeled receptor that specifically binds to an antibody of the invention. Suitable detection techniques that may be applied in concert with the above techniques include autoradiographic detection techniques, detection techniques based on fluorescence, luminescence or phosphorescence or chromogenic detection techniques. These detection techniques are known in the art of detection of biomolecules.

Use may for instance be made of signal producing systems, involving one or more components, at least one component being a detectable label, which generate a detectable signal that relates to the amount of bound and/or unbound label, i.e. the amount of label bound or not bound to the compound being detected. The label is any molecule that produces or can be induced to produce a signal, and preferably is a fluorescer, radio-label, enzyme, chemiluminescer or photosensitizer. Thus, the signal is detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

Suitable labels include, by way of illustration and not limitation, enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH") and horseradish peroxidase; ribozyme; a substrate for a replicase such as QB replicase; promoters; dyes; fluorescers, such as fluorescein, rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine; chemiluminescers such as isoluminol; sensitizers; coenzymes; enzyme substrates; radiolabels such as ¹²⁵I, ¹⁴O, ³H, ⁵⁷Co and ⁷⁵Se; particles such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labeled with a dye, catalyst or other detectable group. Suitable enzymes and coenzymes are disclosed in U.S. Patent No. 4,275,149; U.S. Patent No. 4,318,980; suitable fluorescers and chemiluminescers are disclosed i.a. in U.S. Patent No. 4,275,149.

There are numerous methods by which the label can produce a signal detectable by external means, for example, desirably by visual examination or by electromagnetic radiation, heat, and chemical reagents.

The label or other signal producing system component can also be bound to a specific binding partner, another molecule or to a support.

The label can directly produce a signal, and therefore, additional components are not required to produce a signal. Numerous organic molecules, for example fluorescers, are able to absorb ultraviolet and visible light, where the light absorption transfers energy to these molecules and elevates them to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength. Other labels that directly produce a signal include radioactive isotopes and dyes. Alternately, the label may need other components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal, which may include substrates, coenzymes, enhancers, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances. A detailed discussion of suitable signal producing systems can be found in U.S. Patent No. 5,185,243.

The label can be bound covalently to numerous specific binding partners: an antibody; a receptor for an antibody; a receptor that is capable of binding to a small molecule conjugated to an antibody; or a ligand analog. Bonding of the label to the specific binding partner may be accomplished by chemical reactions which result in replacing a hydrogen atom of the label with a bond to the specific binding partner member or may include a linking group between the label and the specific binding partner. Other signal producing system components may also be bound covalently to specific binding partners. For example, two signal producing system components such as a fluorescer and quencher can each be bound to a different antibody that forms a specific complex with the analyte. Formation of the complex brings the fluorescer and quencher in close proximity, thus permitting the quencher to interact with the fluorescer to produce a signal. Methods of conjugation are well known in the art. See for example, U.S. Patent No. 3,817,837. This invention also contemplates having an antibody bound to a first signal producing system component and a detectable label as the second signal producing system components. For example, when the detectable label is bound to a ligand analog, the extent of binding of the antibody to the analog can be measured by detecting the signal produced by the interaction of the signal producing system components.

Methods and means provided herein are particularly useful in a diagnostic kit for diagnosing a disease by immunological techniques. Such kits or assays may for example comprise one or more reference markers, one or more reference samples and/or one or more antibodies for any of the markers for the various disease conditions as described herein, and can be used specifically to carry put a method or use according to the present invention, Methods for measuring the expression level of peptides or proteins by MALDI techniques as referred to herein are well known in the art and specific reference is made to the Experimental part described herein below.

The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Example 1. Multiple sclerosis related proteins identified in CSF by advanced mass spectrometry

1. Experimental procedures 1.1. Patient selection

All samples analyzed in this study were taken from patients that were followed prospectively by the Rotterdam Multiple Sclerosis Center and the department of Neurology at Erasmus University Medical Center (Rotterdam, the Netherlands). The samples were all specifically selected from untreated patients. These samples were selected and classified into four groups by an experienced neurologist (RQH). The first designated group consisted of CSF samples from patients suffering from Multiple Sclerosis (MScI, n=44). The second group consisted of samples from patients who were diagnosed with a clinically isolated syndrome of demyelination (CIS, n=40), which is considered as a pre-stage of MScI. The third group of samples was taken from patients with another inflammatory neurological disease (OIND, n=26), to be used as controls. The fourth group, which was also a control group, consisted of samples from patients with some other neurological disease (OND, n=54). All these diagnoses were based on data derived from the examination at the time the CSF sample was taken. Immediately after sampling, the CSF samples were centrifuged to discard cells and cellular elements (10 minutes at 3000 rpm). The samples were subsequently used for routine CSF diagnostics. This included quantification of total protein and high abundant protein (albumin and immunoglobulin G) concentration, assessment of the number of oligoclonal bands, which are present in most, but not all MScI patients, as well as quantification of the intrathecal cell count. The remaining volume of the samples was aliquoted and stored at -80°C, where they remained until sample preparation for this study.

1.2. Sample preparation

Prior to all sample preparation procedures, all samples were blinded. They were subsequently measured and analyzed in a random order. Twenty μl of each CSF sample was put into a 96-microtiter well plate (Nunc. Low binding, VWR, the Netherlands), and an equal amount of 0.2% Rapigest (Waters, USA) in 50 mM ammonium bicarbonate buffer was added to each well. Following a two-minute incubation period at 37°C, 4 μl 0.1 μg/μl gold grade trypsin (Promega, USA) / 3 mM Tris HCl (pH 8.0) was added to each well. The samples were incubated at 37°C for two hours. To obtain a final concentration of 30-50 mM HCl (pH < 2), 2 μl of 500 mM HCl was added. The samples were then incubated for 45 minutes at 37°C, which stopped the digestion reaction.

Following the digestion procedure, the samples were desalted using 96- well zip C18 micro titer plates (Millipore, USA), which had been pre-wetted and washed twice with 100 μl acetonitrile (ACN) per well. The samples were centrifuged (Multifuge 3 S-R, Goffin Meyvis, the Netherlands) at 2000 rpm for five minutes. After the washing step, 3 μl ACN was put on the C 18 resin to prevent drying. Each trypsin digested CSF sample was mixed with 200 μl HPLC grade water / 0.1% trifluoroacetic acid (TFA). The samples were subsequently put onto the washed and pre-wetted 96-well zip C18 plate, and 30 minutes of centrifugation at 1500 rpm was used to load the peptides onto the C 18 material.

The wells were washed twice with 100 μl 0.1% TFA (5 minutes, 1750 rpm). An elution volume of 15 μl 50% ACN/ 0.1% TFA was used to elute the samples in a new 96-well plate. The centrifugation period for the elution step was 30 minutes at 1600 rpm. After this elution step, the samples were stored in 96-well plates, which were covered with aluminum seals, at 4⁰C.

1.3. Measurement A matrix solution was made by dissolving 2 mg α-cyano-4 hydroxy- cinnamic acid (HCCA) in 1 ml ACN, using an ultrasonic bath for 30 minutes. Two μl of elute of each sample was mixed with eight μl of the matrix solution, 0.5 μl of which was spotted onto a MALDI target (600/384 AnchorChipTM with transponder plate, Bruker Daltonics, Germany). All samples were spotted in duplicate. The digestion step and the subsequent measurement in duplicate were performed three times for each sample, resulting in 6 spectra per sample. The samples were all measured using the automated measurement feature of the MALDI-TOF MS (Ultraflex, Bruker Daltonics, Germany). The standard method for peptide measurements (ProteomicsJHPC) was used on the MALDI- TOF MS, with the measurement range set to 500-3400 Dalton (Da).

The following settings were used for the automated measurements: the initial laser power was 25%, and the maximum was 45%. The peak with the highest intensity above the 750 Da had to have a signal-to-noise ratio of at least 5 and a minimum resolution of 5000. Every 50 laser shots the sum spectrum was checked for these criteria. It was rejected if it did not meet these criteria. If 20 sum spectra of 50 laser shots met these criteria, they were combined and saved. If 25 consecutive sum spectra of 50 shots had been rejected, the measurement of the spot was ended and the measurement of the next spot commenced. 1.4. Analysis

The raw binary data files of the MALDI-TOF MS experiments were first converted to ASCII files containing the measured intensities for all channel indices of the spectra. All spectrum files were designated a group number (1 - MScI, 2 - CIS, 3 - OIND, 4 - OND) and serial numbered. To calibrate the channel numbers to masses we used a quadratic fit with a number of internal calibrants, which were five omnipresent tryptic albumin peptides (927.4934, 1226.6051, 1467.8430, 1875.0156 and 2045.0953). The algorithm described by Dekker and co-workers [Dekker, L. J., Boogerd, W., Stockhammer, G., Dalebout, J. et al.,. MoI Cell Proteomics 2005, 4, 1341-1349; Dekker, L. J., Dalebout, J. C, Siccama, L, Jenster. G. et al., Rapid Commun Mass Spectrom 2005, 19, 865-870] was used for peak detection, performed in the statistical language R (http://www.r-project.org). A percentile threshold of 96% was chosen, meaning that the intensity of the peak position must belong to the 4% highest intensity values of the spectrum. The mass window

(minimum distance between two adjacent peak positions) was set at 0.5 Da. After the generation of a peak list for each spectrum using the peak detection algorithm, an analyses matrix was created in R. During this process all peak lists of every sample were checked for the presence of all peaks. Only absence or presence of the peaks was scored. Thus, a peak position that was present in all peak lists of one sample, was designated with a 6 and a peak position present in half of the peak lists was designated with a 3. In this matrix, all samples were tagged with their group number and with a number ranging from 0 to 6 for all separate peak positions. The matrix was subsequently used for statistical analysis of the data. Using a univariate analysis in R, a p-value was determined for every peak position. The Wilcoxon-Mann- Whitney test was used for comparison between the groups [Gaddis, G. M. and Gaddis M. L. Ann Emerg Med. 1990, 19, 1054-9; , Titulaer, M. K., Siccama, I., Dekker, L. J., van Rijswijk, A. L. et al., BMC Bioinformatics 2006, 7, 403.]. For data that does not follow a known theoretical distribution, one can use non-parametric tests. One such test is the Wilcoxon-Mann- Whitney test. Data from multiple groups is combined and the data are tagged so that the group to which they belong is recorded. The combined data set is then sorted according to the value of the data. This type of statistical test is very well suited for the comparison of pre-defined groups, as was the case in these experiments. A cross validation was performed on the same data by randomly assigning a group number to each CSF sample and then repeating the Wilcoxon-Mann- Whitney test. This scrambling procedure was subsequently repeated 10.000 times, which gives an impression of the probability to find a significant differentially expressed peptide by chance.

1.5. Identification

The next step in the process was identification of the differentially expressed peptides. Due to the limitations of the MALDI-TOF strategy, the differentially expressed peptides could not be identified immediately from the complete CSF sample, due to the high amount of peaks per spectrum. So, for identification of the peptides alternative methods were needed. We used two different strategies.

Firstly, we determined the accurate masses of the peptides with a 9.4 Tesla (MALDI) Fourier Transform Mass Spectrometer (FTMS, Bruker

Daltonics, Germany). Due to the excellent mass accuracy and precision of this technique it is suited to determine the masses of the peptides up to an accuracy of better than 1 ppm. Because the HCCA matrix is not compatible with orthogonal MALDI (Apex I, Bruker Daltonics, Germany), we used another matrix molecule, 2,5-dihydroxy benzoic acid (DHB).

Secondly, we used a nano-LC system (Ultimate system, Dionex, USA) to separate the digested CSF peptides on a C18 reversed phase column, followed by off-line MALDI-TOF/TOF mass spectrometry. The digested CSF sample was injected and separated on this column during a 130 min LC run, using a 90 minute gradient of increasing concentrations of ACN (Solvent A: 95% TFA (0.1%) in water, 5% ACN (v/v); Solvent B: 20% TFA (0.1%) in water, 80% ACN), and subsequently spotted on a MALDI target plate. The MALDI target plate used in this nano-LC experiment was a pre-spotted target plate (PAC 384 plate, Bruker Daltonics, Germany). On this plate the matrix was already previously applied and only the sample has to be added. By dividing a single sample into 384 separate spots and corresponding spectra, it proved to be possible to acquire the required mass windows for several of the differentially expressed peptides.

Measurement of the pre-spotted plate was done in an automated way, using the Warp-LC software package, version 1.0 (Bruker Daltonics,

Germany) on the MALDITOF/ TOF, which first generated a mass spectrum for all spots and then proceeded to generate MS/MS spectra, in a data dependent manner, for peaks which were suitable for MS/MS by a series of pre-set conditions, such as, for example, a sufficiently high signal-to-noise ratio. Identifications were obtained by database searches of the MS/MS spectra using Mascot search program, version 2.1 and the SwissProt database (version 51.2), using the standard settings of the Warp-LC software package. The score threshold for peptide identification was a Mascot score of over 54, which correlates with a p-value < 0.05. Identifications were confirmed by determination of accurate mass by MALDI-FTMS. In a separate effort to identify the differentially expressed peptides we also used an Orbitrap mass spectrometer with electrospray ionization (Thermo Electron, Germany), using a C 18 column nano-LC system online connected to the device. Five microliter of the digested CSF was injected into the nano-LC and subsequently the digested peptides were separated on the C 18 column. After ionization the peptides were measured in the Orbitrap, using a data-dependent acquisition mode for the MS/MS identification step, which occurred in the ion trap. The MS/MS identifications were obtained using the Bioworks 3.2 software package (Thermo Fisher Scientific, Germany) and its' SEQUEST feature, using the standard settings, with XC scores of 1.5, 2.0 and 2.5 for single, double and triple charged ions respectively. With this method we also used the SwissProt database for database searching as mentioned above.

1.6. Immunoassay As specified by the manufacturer (DakoCytomation, Denmark), we used a commercially available ELISA kit for chromogranin A to determine the chromogranin A concentrations in the CSF samples. The sample volumes available were not sufficient to do this ELISA for all samples. Hundred twentythree samples of the original test set were evaluated (32 MScI, 32 CIS, 25 OIND and 34 OND samples). For external validation, we had available a second set of CSF samples of MScI patients (n = 19) and OND controls (n = 18), including non neurological cancer, intracranial hypertension, Sneddon syndrome and headache.

2. Results

2.1. Clinical information

Of the 44 patients in the MScI group, 30 patients had relapsing remitting (RR) MScI and 14 had primary progressive (PP) MScI (Table 1). The mean age of the patients in this group was 42.1 ± 11.3 years, and the mean protein concentration of the CSF was 0.41 ± 0.13 g/1. The group contained 12 males and 32 females. The median of the time that these patients had been afflicted with MScI was nearly two years.

Table 1. Clinical information, including routine protein quantifications, of all patients included in this study.

# Excepting two patients (72 and 27 months), all CIS samples were taken within twenty months after the first symptoms were observed.

- Concentrations: p>0.05 for all comparisons (two-tailed t-test), except total protein concentration for CIS-OIND (p=0.026) and IgG concentration for MScI-OND (p=0.035).

The CIS group (n=40) consisted of 12 males and 28 females, nearly

70 percent of whom had been diagnosed with optic neuritis (Table 1). The other patients were diagnosed with one, or a combination, of the other possible localizations of a CIS, like for example brainstem syndromes or myelitis. The mean age of this second group was 33.7 ± 9.4 years, and the mean protein concentration of the CSF was 0.36 ± 0.11 g/l. The median time between the sampling of the CSF and the occurrence of the first symptoms in these patients was three months.

The OIND group (n=26) contained 7 males and 19 females, with a mean age of 49.6 ± 16.3 years. The mean CSF protein concentration was 0.43 ± 0.16 g/l (Table 1). Diagnoses of the patients in this group were bacterial and sterile infections (n=17), vasculitis (n=7), and Guillain Barre Syndrome (n=2). The 54 patients in the OND group had a mean age of 48.1 ± 16.7 years. This group contained 23 males and 31 females, and the CSF samples had a mean protein concentration of 0.42 ± 0.22 g/1 (Table 1). The diagnoses set for these patients were headaches (n=19), neurological degeneration (n=ll), nonneurological disease (n=10), infarct (n=8), brain tumor (n=4), and other (n=2)

The total protein concentrations of the CSF samples did not differ significantly between the groups when subjected to a two-tailed t-test. Also, the albumin and immunoglobulin G (IgG) concentrations of the CSF samples did not differ significantly between the groups.

2.2. Peak detection and data analysis

An average of 515 peaks was detected per MALDI-TOF spectrum. After spectrum conversion, the matrix was created, which consisted of a total of 1755 peaks. The significance of difference in distribution over the four groups was tested for each peak.

The significant difference between the MScI group and the OND group (p<0.05: 132 peaks; p<0.01: 44 peaks) indicates obvious differences in the CSF of MScI patients compared to that of the non-inflammatory control patients. The obvious skew towards the low p-values is clearly visible in the p- value histogram (figure 1). The largest statistical difference (figure 2) was observed in the comparison of the CIS group and the OND group (p<0.05: 161 peaks; p<0.01: 60 peaks).

The comparison of the MScI group with the OIND group (figure 3), and also that of the CIS group with the OIND group (figure 4), shows a number of peak positions with a statistically significant p-value (p<0.05: 127 peaks; p<0.01:12 peaks, and p<0.05: 134 peaks; p<0.01: 27 peaks, respectively). Both these p-value histograms are also skewed towards the lower p-values, and, in this region, the height of the bars clearly exceeds the height of the red line, which reflects the expected statistical background. Comparison of the MScI group with the CIS group (figure 5) shows few peaks with a significant p-value (p<0.05: 50 peaks; p<0.01: 10 peaks). The height of the bars in the low p-value area clearly lies below the red line generated by the randomized comparison, indicating that there is no significant difference between the CSF samples of the patients classified in the MScI group compared to the patients in the CIS group. The comparison between the two control groups (OIND vs. OND, figure 6) shows only a slightly higher number of statistically significant peak positions (p<0.05: 101 peaks; p<0.01: 12 peaks). The height of the bars in the low p-value area lies at about the same height as the red line generated by the randomized comparison, which indicates that these two groups are not statistically significantly different.

2.3. Identification Using the combination of the FTMS and the nano-LC MALDI-

TOF/TOF techniques we were able to determine the amino acid sequence of three of the differentially expressed peptides in the comparison between MScI and OND.

These peptides were identified to be tryptic peptides of chromogranin A, clusterin and complement C3 by offline nano-LC MALDI- TOF/TOF. Determination of the accurate mass of these three tryptic peptides by FTMS resulted in parent ionmasses differing 0.19, 0.27 and 0.67 ppm from the theoretical masses for those specific peptides of chromogranin A, clusterin and complement C3, respectively. Although the Mascot score for the clusterin peptide is below 54, the additional accurate mass obtained by FTMS and the XC score (3.22, doubly charged) obtained for this peptide validated this identification in separate ways.

Using the sequencing capabilities of the Orbitrap mass spectrometer we were able to identify 10 peptides from the list of differentiating peptide peaks (Table 2 and 3). The accurate masses of the parent ions of these identified peptides were further confirmed by FTMS. All identified peptides showed accuracy below 1 ppm for FTMS (Table 2). The additional peptides identified for each protein using the Orbitrap set-up were not detected using the MALDI-TOF profiling approach and thus they were not present in the comparison matrix.

Table 2. Identified differentially expressed peptides and corresponding proteins (p<0.01) amongst the patient groups.

* Number of peptides in the direct (without nano-LC fractionation) MALDI comparison was 1.

- Column 1: Bold denotes group with elevated expression for that specific protein

- 1 missed cleavage allowed

Table 3: Sequence coverage and scores of significantly differentially expressed peptides found by MALDI-MS.

- Not found.

* Using SwissProtein database (October 2006).

^HXC value in agreement with the HUPO criteria (XC = 1.8, 2.5 and 3.1 for single, double and triple charged ions respectively). x Other peptide(s) of this protein were identified with XC values in agreement with the HUPO criteria.

Testing of chromogranin A levels by ELISA showed that MScI samples had higher chromogranin A concentrations than non-inflammatory control samples (table 4A) which was in agreement with the proteomics analysis. The difference in concentration was statistically significant (p = 0.00034). No statistically significant differences were observed in the comparisons of the chromogranin A concentrations of the other groups. The external validation in a new set of samples showed that also these MScI samples contained significantly higher chromogranin A concentrations compared with the non-inflammatory controls (table 4B). Table 4A.

A: Chromogranin A concentrations were determined in the original test set by means of a commercially available ELISA. A total of 123 CSF samples (32 MScI, 34 OND, 32 CIS and 25 OIND) was tested. The Chromogranin A concentration was significantly higher (p = 0.00034) in MScI than in OND, using a t-test. The concentrations of Chromogranin A in the other comparisons did not yield statistically significant p-values.

Table 4B.

Chromogranin A concentrations were also determined in an external validation set of CSF MScI and OND samples. A total of 37 samples'were tested (19 MScI and 18 OND). A t-test showed a statistically significant p-value (p = 0.02) for this comparison.

3. Conclusion

A main observation of this study is that, on the basis of proteomic CSF analysis, the group of MScI patients could be clearly distinguished from the noninflammatory neurological controls. However, as MScI is considered to be an inflammatory disease, it was imperative to include a group of controls covering other CNS inflammatory neurological diseases. We were also able to clearly differentiate between CSF peptide profiles of the MScI patients and inflammatory neurological controls. As MScI has both an inflammatory and a neurodegenerative component, comparison with both control groups is essential to ensure that the control groups covered both the inflammatory and the non-inflammatory phase of the disease.

We were also able to make similar distinctions in the proteomic CSF analyses of the CIS patients when compared to the non-inflammatory controls, as well as in the comparison of the CIS patients with the inflammatory neurological controls. The fact that the MScI and CIS groups behaved similarly fits with the clinical observations that, in the majority of the cases, CIS is a prestage of MScI. It can be assumed that similar immunopathological and neurodegenerative processes take place in both conditions. Another indication of the similarities between the proteomic profiles of the CSF samples of the patients in the MScI group and those of the CIS group is the overlap of the peptides found in the comparisons of MScI with the non-inflammatory controls (OND) and the comparison of CIS with OND. Eleven of the forty-four peptide peaks (25%) found to be significantly (p<0.01) different in the comparison of

MScI and OND was also found to be significantly different in the comparison of CIS and OND. If the significance cut-off is set to p<0.05, this overlap increases to nearly 38%.

We also compared two major distinct disease courses of MScI, PP MScI and RR MScI. Specifically for this sub -comparison we had included a relatively high number of PP MScI patients in this study (32% of the MScI samples (normal 10-15% of the total MScI population)). However, we did not observe any significant differences between PP MScI and RR MScI (data not shown). Because pathology changes over time in MScI [21, 22, 23, 24], one could imagine differences in CSF proteomic profiles as the disease duration progresses. The MScI samples were divided into a short and a long disease duration group, according to their position in relation to the median time between first symptoms and CSF sampling. No significant differences were observed in this comparison, compared to the statistical background (data not shown).

It has been suggested that the course of the disease is more benign in MScI patients without oligoclonal Immunoglobulin G bands in CSF. One might therefore expect to find other differences in CSF composition when comparing MScI patients with oligoclonal Immunoglobulin G bands in the CSF versus MScI patients without these bands. However, we did not observe this. Finally, also no differences were observed between males and females, compared to the statistical background (data not shown). These sub-analyses lend support to the interpretation that common peptide patterns are shared within the whole group of MScI patients.

It has been shown that factors such as sample stability and a low number of measurements per sample can cause difficulties regarding the reproducibility of proteomic profiling studies. Other studies have shown the low reproducibility of peak height in MALDI-TOF MS. The method described here is less affected by these variations, as the heights of the peaks were not included in this analysis because quantitative measurements of peak heights with MALDI TOF MS are poorly reproducible, with standard deviations up to 30%. In this study only the absence or presence of the peaks was scored. Considering the fact that these data are a summation of three separate MALDI-TOF experiments, in which CSF samples were measured in duplicate, we also performed three separate analyses for these three experiments. A similar p value diagram for each experiment was obtained, compared to the combined experiments. Similarly, the differentially expressed peptides that were found in the total analysis also had low p-values in the separate analyses, showing that these results were reproducible inter assay wise.

Another beneficial factor is the lower protein load, especially of high abundant proteins such as albumin in CSF, which allows CSF peptide profiling without complicated sample pre-treatment steps (e.g. high-abundant protein depletion) that are essential to serum peptide profiling. Other studies have already delved into the CSF proteomic profile. Although these studies identified a number of interesting proteins that were present in CSF of MScI patients, these analyses were performed in a setting with limited numbers of patients. It is hard to compare these studies, as the set-up and objectives have been very different. The studies mentioned aimed to identify as many proteins as possible in CSF of MScI patients, whereas we aimed to pinpoint peptide distinctions between MScI patients and controls. The method used in our study also differed from these studies in the fact that our sample pre-treatment was much less complex, as we did not perform a two-dimensional gel electrophoresis procedure prior to the trypsin digestion. Another difference is that the number of samples we analyzed is considerably higher, which allowed for more advanced statistics during the analysis.

In total, we identified eleven proteins in association with MScI (Table 2). Five of them, neuronal pentraxin receptor, contactin 1, beta V spectrin, RNA binding motif protein 7 and osteopontin, have not been reported in association with MScI previously. These proteins may help to reveal new pathways associated with MScI. Neuronal pentraxin receptor is a molecule implied in synapse formation and neuronal remodeling. It is also suggested to be involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris. Contactins mediate cell surface interactions during nervous system development. They are involved in the formation of paranodal axo-glial junctions in myelinated peripheral nerves and in the signaling between axons and myelinating glial cells. Beta V spectrin is expressed at very low levels in many tissues, with strongest expression in, among others, cerebellum and spinal cord. It is now well recognized that spectrins may contribute to the establishment and maintenance of membrane order.

The other six proteins, apolipoprotein D, chromogranin A, clusterin, complement C3, complement C4A and complement C4B have been previously reported in association with MScI. Apolipoprotein D, which is involved in the removal of lipids during nerve cell degeneration, has been reported previously to be present in elevated levels in MScI.

The relevance for the role of complement in MScI pathogenesis is a matter of a long debate, with several papers suggesting elevated levels in affected tissue and in CSF. This detection of several complement components in the CSF of MScI patients lends further support to the role of complement in this disease.

The association of CSF clusterin, also known as apolipoprotein J, with MScI is of particular interest in this respect, because this molecule serves as a regulator (inhibitor) of complement activity. In addition, it is considered as an important factor in neuronal cell survival. There is an earlier report that suggested elevated CSF clusterin levels in patients with central nervous system inflammation [Polihronis, M., Paizis, K., Carter, G., Sedal, L. et al., J Neurol Sci 1993, 115, 230-233].

Finally, the detection of chromogranin A in association with MScI is interesting. Although the neurodegenerative component of MScI pathology has remained an enigma, an important role of innate immune cells and signals is obvious. It is of interest that chromogranin A can induce neurotoxicity in microglial cells [Taylor, D. L., Diemel, L. T., Cuzner, M. L., and Pocock, J. M. J Neurochem 2002, 85, 1179-91]. Chromogranin A is not differentially expressed in the comparison between MScI and OIND, so this protein could also be a marker for non-specific inflammation. Other reports have shown that this protein is elevated in other neurodegenerative diseases such as Alzheimer's disease [Taylor, D. L., Diemel, L. T., Cuzner, M. L., and Pocock, J. M. J Neurochem 2002, 85, 1179-91, Lechner, T., Adlassnig, C, Humpel, C, Kaufmann, W. A., et al.,. Exp Gerontol 2004, 39, 101-13], which may indicate that chromogranin A is involved in the neurodegenerative component of MScI. While proteins and peptides identified by this shotgun approach may certainly be specific for MScI, the possibility of non-MScl pathology specific differentially expressed proteins cannot be ruled out. While non-specific differentially expressed proteins can also add valuable information about disease processes, more research on chromogranin A could provide a better understanding about the role of this protein in MScI disease processes. So, for example, it will be of interest to determine whether high CSF chromogranin A levels are associated with more aggressive clinical disease types.

In conclusion, the MALDI-TOF analysis of tryptic digested CSF proteins showed significant differences between MScI patients and control patients (MScI vs. OND: 44 peptides with p<0.01), as well as significant differences between CIS patients and control patients (CIS vs. OND: 60 peptides with p<0.01). Among these proteins were important factors in complement mediated inflammatory reaction, such as complement C3, complement C4A, complement C4B and clusterin, as well as several proteins that were not previously reported to be differentially expressed in MScI and its' pre-stage (CIS). These new potential markers include functionally interesting proteins such as chromogranin A, neuronal pentraxin receptor and contactin 1.

SEQUENCE LISTING

<110> Erasmus University Medical Center Rotterdam

<120> Peptide markers for diagnosis of neurodegenerative disease

<130> P83502PC00

<150> PCT/2007/050014 <151> 2007-01-12

<160> 23

<170> Patentln version 3.3

<210> 1

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Chromogranin A

<400> 1

Tyr Pro GIy Pro GIn Ala GIu GIy Asp Ser GIu GIy Leu Ser GIn GIy 1 5 10 15

Leu VaI Asp Arg 20

<210> 2

<211> 10

<212> PRT <213> Artificial sequence

<220>

<223> Clusterin <400> 2

Thr Leu Leu Ser Asn Leu GIu GIu Ala Lys 1 5 10

<210> 3

<211> 14

<212> PRT <213> Artificial sequence

<220>

<223> Complement C3

<400> 3

Ala GIy Asp Phe Leu GIu Ala Asn Tyr Met Asn Leu GIn Arg 1 5 10

<210> 4

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Complement C4B

<400> 4

Ala Ser Ala GIy Leu Leu GIy Ala His Ala Ala Ala lie Thr Ala Tyr 1 ' 5 10 ^' 15

Ala Leu Thr Leu Thr Lys 20

<210> 5 <211> 16 <212> PRT <213> Artificial sequence

<220>

<223> Beta V spectrin

<400> 5

Trp lie Asn Asn VaI Phe GIn Cys GIy GIn Ala GIy He Lys He Arg 1 5 10 15

<210> 6

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Hypothetical protein XP_011125

<400> 6

He Ser His GIu Leu Asp Ser Ala Ser Ser GIu VaI Asn 1 5 10

<210> 7 <211> 7

<212> PRT

<213> Artificial sequence

<220> <223> Apolipoprotein D

<400> 7

VaI Leu Asn GIn GIu Leu Arg i 5

<210> 8 <211> 9 <212> PRT

<213> Artificial sequence

<220> <223> Complement C4A

<400> 8

GIy Leu GIn Asp GIu Asp GIy Tyr Arg 1 5

<210> 9

<211> 11

<212> PRT <213> Artificial sequence

<220>

<223> Contactin 1

<400> 9

VaI Gin VaI Thr Ser GIn GIu Tyr Ser Ala Arg 1 5 10

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Neuronal pentraxin receptor

<400> 10

GIn Thr Ala Leu GIn GIn GIu Ala Arg 1 5

<210> 11 <211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> RNA binding motif protein 7

<400> 11

VaI Thr GIu GIu Leu Leu Phe GIu Leu Phe His GIn Ala GIy Pro VaI 1 5 10 15

lie Lys VaI Lys 20

<210> 12

<211> 457

<212> PRT

<213> Artificial sequence

<220>

<223> chromogranin A

<400> 12

Met Arg Ser Ala Ala VaI Leu Ala Leu Leu Leu Cys Ala GIy GIn VaI 1 5 10 15

Thr Ala Leu Pro VaI Asn Ser Pro Met Asn Lys GIy Asp Thr GIu VaI 20 25 30

Met Lys Cys lie VaI GIu VaI lie Ser Asp Thr Leu Ser Lys Pro Ser 35 40 45

Pro Met Pro VaI Ser GIn GIu Cys Phe GIu Thr Leu Arg GIy Asp GIu 50 55 60

Arg lie Leu Ser lie Leu Arg His GIn Asn Leu Leu Lys GIu Leu GIn 65 70 75 80

Asp Leu Ala Leu GIn GIy Ala Lys GIu Arg Ala His GIn GIn Lys Lys 85 90 95

His Ser GIy Phe GIu Asp GIu Leu Ser GIu VaI Leu GIu Asn GIn Ser 100 105 110

Ser GIn Ala GIu Leu Lys GIu Ala VaI GIu GIu Pro Ser Ser Lys Asp 115 120 125

VaI Met GIu Lys Arg GIu Asp Ser Lys GIu Ala GIu Lys Ser GIy GIu 130 135 140

Ala Thr Asp GIy Ala Arg Pro GIn Ala Leu Pro GIu Pro Met GIn GIu 145 150 155 160

Ser Lys Ala GIu GIy Asn Asn GIn Ala Pro GIy GIu GIu GIu GIu GIu 165 170 175

GIu GIu GIu Ala Thr Asn Thr His Pro Pro Ala Ser Leu Pro Ser GIn 180 185 190

Lys Tyr Pro GIy Pro GIn Ala GIu GIy Asp Ser GIu GIy Leu Ser GIn 195 200 205

GIy Leu VaI Asp Arg GIu Lys GIy Leu Ser Ala GIu Pro GIy Trp GIn 210 215 220

Ala Lys Arg GIu GIu GIu GIu GIu GIu GIu GIu GIu Ala GIu Ala GIy 225 230 235 240

GIu GIu Ala VaI Pro GIu GIu GIu GIy Pro Thr VaI VaI Leu Asn Pro 245 250 255

His Pro Ser Leu GIy Tyr Lys GIu lie Arg Lys GIy GIu Ser Arg Ser 260 265 270 GIu Ala Leu Λla VaI Asp GIy Ala GIy Lys Pro GIy Ala GIu GIu Ala 275 280 285

GIn Asp Pro GIu GIy Lys GIy GIu GIn GIu His Ser GIn GIn Lys GIu 290 295 300

GIu GIu GIu GIu Met Ala VaI VaI Pro GIn GIy Leu Phe Arg GIy GIy 305 310 315 320

Lys Ser GIy GIu Leu GIu GIn GIu GIu GIu Arg Leu Ser Lys GIu Trp

325 330 335

GIu Asp Ser Lys Arg Trp Ser Lys Met Asp GIn Leu Ala Lys GIu Leu 340 345 350

Thr Ala GIu Lys Arg Leu GIu GIy GIn GIu GIu GIu GIu Asp Asn Arg 355 360 365

Asp Ser Ser Met Lys Leu Ser Phe Arg Ala Arg Ala Tyr GIy Phe Arg 370 375 380

GIy Pro GIy Pro GIn Leu Arg Arg GIy Trp Arg Pro Ser Ser Trp GIu 385 390 395 400

Asp Ser Leu GIu Ala GIy Leu Pro Leu GIn VaI Arg GIy Tyr Pro GIu

405 410 415

GIu Lys Lys GIu GIu GIu GIy Ser Ala Asn Arg Arg Pro GIu Asp GIn 420 425 430

GIu Leu GIu Ser Leu Ser Ala lie GIu Ala GIu Leu GIu Lys VaI Ala 435 440 445

His GIn Leu GIn Ala Leu Arg Arg GIy 450 455

<210> 13 <211> 501 <212> PRT

<213> Artificial sequence

<220> <223> clusterin

<400> 13

Met GIn VaI Cys Ser GIn Pro GIn Arg GIy Cys VaI Arg GIu GIn Ser 1 5 10 15

Ala lie Asn Thr Ala Pro Pro Ser Ala His Asn Ala Ala Ser Pro GIy 20 25 30

GIy Ala Arg GIy His Arg VaI Pro Leu Thr GIu Ala Cys Lys Asp Ser 35 40 45

Arg lie GIy GIy Met Met Lys Thr Leu Leu Leu Phe VaI GIy Leu Leu 50 55 60

Leu Thr Trp GIu Ser GIy GIn VaI Leu GIy Asp GIn Thr VaI Ser Asp 65 70 75 80

Asn GIu Leu Gin GIu Met Ser Asn GIn GIy Ser Lys Tyr VaI Asn Lys 85 90 95

GIu lie GIn Asn Ala VaI Asn GIy VaI Lys GIn lie Lys Thr Leu lie 100 105 110

GIu Lys Thr Asn GIu GIu Arg Lys Thr Leu Leu Ser Asn Leu GIu GIu 115 120 125

Ala Lys Lys Lys Lys GIu Asp Ala Leu Asn GIu Thr Arg GIu Ser GIu 130 135 140

Thr Lys Leu Lys GIu Leu Pro GIy VaI Cys Asn GIu Thr Met Met Ala 145 150 155 160 Leu Trp GIu GIu Cys Lys Pro Cys Leu Lys Gin Thr Cys Met Lys Phe 165 170 175

Tyr Ala Arg VaI Cys Arg Ser GIy Ser GIy Leu VaI GIy Arg GIn Leu 180 185 190

GIu GIu Phe Leu Asn GIn Ser Ser Pro Phe Tyr Phe Trp Met Asn GIy 195 200 205

Asp Arg lie Asp Ser Leu Leu GIu Asn Asp Arg GIn GIn Thr His Met 210 215 220

Leu Asp VaI Met GIn Asp His Phe Ser Arg Ala Ser Ser lie lie Asp 225 230 235 240

GIu Leu Phe Gin Asp Arg Phe Phe Thr Arg GIu Pro GIn Asp Thr Tyr 245 250 255

His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe 260 265 270

Pro Lys Ser Arg lie VaI Arg Ser Leu Met Pro Phe Ser Pro Tyr GIu 275 280 285

Pro Leu Asn Phe His Ala Met Phe GIn Pro Phe Leu GIu Met lie His 290 295 300

GIu Ala GIn GIn Ala Met Asp He His Phe His Ser Pro Ala Phe GIn 305 310 315 320

His Pro Pro Thr GIu Phe lie Arg GIu GIy Asp Asp Asp Arg Thr VaI 325 330 335

Cys Arg GIu lie Arg His Asn Ser Thr GIy Cys Leu Arg Met Lys Asp 340 345 350

GIn Cys Asp Lys Cys Arg GIu He Leu Ser VaI Asp Cys Ser Thr Asn 355 360 365 Asn Pro Ser GIn Ala Lys Leu Arg Arg GIu Leu Asp GIu Ser Leu GIn 370 375 380

VaI Ala GIu Arg Leu Thr Arg Lys Tyr Asn GIu Leu Leu Lys Ser Tyr 385 390 395 400

GIn Trp Lys Met Leu Asn Thr Ser Ser Leu Leu GIu GIn Leu Asn GIu 405 410 415

GIn Phe Asn Trp VaI Ser Arg Leu Ala Asn Leu Thr GIn GIy GIu Asp 420 425 430

GIn Tyr Tyr Leu Arg VaI Thr Thr VaI Ala Ser His Thr Ser Asp Ser 435 440 445

Asp VaI Pro Ser GIy VaI Thr GIu VaI VaI VaI Lys Leu Phe Asp Ser 450 455 460

Asp Pro He Thr VaI Thr VaI Pro VaI GIu VaI Ser Arg Lys Asn Pro 465 470 475 480

Lys Phe Met GIu Thr VaI Ala GIu Lys Ala Leu GIn GIu Tyr Arg Lys 485 490 495

Lys His Arg GIu GIu 500

<210> 14 <211> 1288

<212> PRT

<213> Artificial sequence

<220> <223> complement component 3

<400> 14 Met GIy Pro Thr Ser GJy Pro Ser Leu Lsu Leu Leu Leu Leu Tbr His 1 5 10 15

Leu Pro Leu Ala Leu GIy Ser Pro Met Tyr Ser lie lie Thr Pro Asn 20 25 30

lie Leu Arg Leu GIu Ser GIu GIu Thr Met VaI Leu GIu Ala His Asp 35 40 45

Ala GIn GIy Asp VaI Pro VaI Thr VaI Thr VaI His Asp Phe Pro GIy 50 55 60

Lys Lys Leu VaI Leu Ser Ser GIu Lys Thr VaI Leu Thr Pro Ala Thr 65 70 75 80

Asn His Met GIy Asn VaI Thr Phe Thr lie Pro Ala Asn Arg GIu Phe 85 90 95

Lys Ser GIu Lys GIy Arg Asn Lys Phe VaI Thr VaI GIn Ala Thr Phe 100 105 110

GIy Thr GIn VaI VaI GIu Lys VaI VaI Leu VaI Ser Leu GIn Ser GIy 115 120 125

Tyr Leu Phe lie GIn Thr Asp Lys Thr lie Tyr Thr Pro GIy Ser Thr 130 135 140

VaI Leu Tyr Arg lie Phe Thr VaI Asn His Lys Leu Leu Pro VaI GIy 145 150 155 160

Arg Thr VaI Met VaI Asn He GIu Asn Pro GIu GIy He Pro VaI Lys 165 170 175

Gin Asp Ser Leu Ser Ser GIn Asn GIn Leu GIy VaI Leu Pro Leu Ser 180 185 190

Trp Asp He Pro GIu Leu VaI Asn Met GIy GIn Trp Lys He Arg Ala 195 200 205 Tyr Tyr GIu Asn Ser Pro GIn GIn VaI Phe Ser Thr GIu Phe GIu VaI 210 215 220

Lys GIu Tyr VaI Leu Pro Ser Phe GIu VaI lie VaI GIu Pro Thr GIu 225 230 235 240

Lys Phe Tyr Tyr lie Tyr Asn GIu Lys GIy Leu GIu VaI Thr lie Thr 245 250 255

Ala Arg Phe Leu Tyr GIy Lys Lys VaI GIu GIy Thr Ala Phe VaI lie 260 265 270

Phe GIy lie GIn Asp GIy GIu GIn Arg lie Ser Leu Pro GIu Ser Leu 275 280 285

Lys Arg He Pro lie GIu Asp GIy Ser GIy GIu VaI VaI Leu Ser Arg 290 295 300

Lys VaI Leu Leu Asp GIy VaI GIn Asn Pro Arg Ala GIu Asp Leu VaI 305 310 315 320

GIy Lys Ser Leu Tyr VaI Ser Ala Thr VaI He Leu His Ser GIy Ser 325 330 335

Asp Met VaI GIn Ala GIu Arg Ser GIy He Pro He VaI Thr Ser Pro 340 345 350

Tyr GIn He His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro GIy Met 355 360 365

Pro Phe Asp Leu Met VaI Phe VaI Thr Asn Pro Asp GIy Ser Pro Ala

370 375 380

Tyr Arg VaI Pro VaI Ala VaI GIn GIy GIu Asp Thr VaI GIn Ser Leu

385 390 395 400

Thr GIn GIy Asp GIy VaI Ala Lys Leu Ser He Asn Thr His Pro Ser 405 410 415

GIn Lys Pro Leu Ser lie Thr VaI Arg Thr Lys Lys GIn GIu Leu Ser 420 425 430

GIu Ala GIu GIn Ala Thr Arg Thr Met GIn Ala Leu Pro Tyr Ser Thr 435 440 445

VaI GIy Asn Ser Asn Asn Tyr Leu His Leu Ser VaI Leu Arg Thr GIu 450 455 460

Leu Arg Pro GIy GIu Thr Leu Asn VaI Asn Phe Leu Leu Arg Met Asp 465 470 475 480

Arg Ala His GIu Ala Lys lie Arg Tyr Tyr Thr Tyr Leu lie Met Asn

485 490 495

Lys GIy Arg Leu Leu Lys Ala GIy Arg GIn VaI Arg GIu Pro GIy GIn 500 505 510

Asp Leu VaI VaI Leu Pro Leu Ser lie Thr Thr Asp Phe lie Pro Ser 515 520 525

Phe Arg Leu VaI Ala Tyr Tyr Thr Leu lie GIy Ala Ser GIy GIn Arg 530 535 540

GIu VaI VaI Ala Asp Ser VaI Trp VaI Asp VaI Lys Asp Ser Cys VaI 545 550 555 560

GIy Ser Leu VaI VaI Lys Ser GIy GIn Ser GIu Asp Arg GIn Pro VaI

565 570 575

Pro GIy GIn GIn Met Thr Leu Lys lie GIu GIy Asp His GIy Ala Arg 580 585 590

VaI VaI Leu VaI Ala VaI Asp Lys GIy VaI Phe VaI Leu Asn Lys Lys 595 600 605 Asn Lys Leu Thr GIn Ser Lys lie Trp Asp VaI VaI GIu Lys Ala Asp 610 615 620

lie GIy Cys Thr Pro GIy Ser GIy Lys Asp Tyr Ala GIy VaI Phe Ser 625 630 635 640

Asp Ala GIy Leu Thr Phe Thr Ser Ser Ser GIy GIn GIn Thr Ala GIn 645 650 655

Arg Ala GIu Leu GIn Cys Pro GIn Pro Ala Ala Arg Arg Arg .Arg Ser 660 665 670

VaI GIn Leu Thr GIu Lys Arg Met Asp Lys VaI GIy Lys Tyr Pro Lys 675 680 685

GIu Leu Arg Lys Cys Cys GIu Asp GIy Met Arg GIu Asn Pro Met Arg 690 695 700

Phe Ser Cys GIn Arg Arg Thr Arg Phe lie Ser Leu GIy GIu Ala Cys 705 710 715 720

Lys Lys VaI Phe Leu Asp Cys Cys Asn Tyr lie Thr GIu Leu Arg Arg 725 730 735

GIn His Ala Arg Ala Ser His Leu GIy Leu Ala Arg Ser Asn Leu Asp 740 745 750

GIu Asp lie lie Ala GIu GIu Asn lie VaI Ser Arg Ser GIu Phe Pro 755 760 765

GIu Ser Trp Leu Trp Asn VaI GIu Asp Leu Lys GIu Pro Pro Lys Asn 770 775 780

GIy lie Ser Thr Lys Leu Met Asn lie Phe Leu Lys Asp Ser He Thr 785 790 795 800

Thr Trp GIu lie Leu Ala VaI Ser Met Ser Asp Lys Lys GIy He Cys 805 810 815 VaI Ala Asp Pro Phe GIu VaI Thr VaI Met GIn Asp Phe Phe He Asp 820 825 830

Leu Arg Leu Pro Tyr Ser VaI VaI Arg Asn GIu GIn VaI GIu He Arg 835 840 845

Ala VaI Leu Tyr Asn Tyr Arg GIn Asn GIn GIu Leu Lys VaI Arg VaI 850 855 860

GIu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg 865 870 875 880

Arg His GIn GIn Thr VaI Thr He Pro Pro Lys Ser Ser Leu Ser VaI 885 890 895

Pro Tyr VaI He VaI Pro Leu Lys Thr GIy Leu GIn GIu VaI GIu VaI 900 905 910

Lys Ala Ala VaI Tyr His His Phe He Ser Asp GIy VaI Arg Lys Ser 915 920 925

Leu Lys VaI VaI Pro GIu GIy He Arg Met Asn Lys Thr VaI Ala VaI 930 935 940

Arg Thr Leu Asp Pro GIu Arg Leu GIy Arg GIu GIy VaI GIn Lys GIu 945 950 955 960

Asp He Pro Pro Ala Asp Leu Ser Asp GIn VaI Pro Asp Thr GIu Ser 965 970 975

GIu Thr Arg He Leu Leu GIn GIy Thr Pro VaI Ala GIn Met Thr GIu 980 985 990

Asp Ala VaI Asp Ala GIu Arg Leu Lys His Leu He VaI Thr Pro Ser 995 1000 1005

GIy Cys GIy GIu GIn Asn Met He GIy Met Thr Pro Thr VaI He W

56

1010 1015 1020

Ala VaI His Tyr Leu Asp GIu Thr GIu GIn Trp GIu Lys Phe GIy 1025 1030 1035

Leu GIu Lys Arg GIn GIy Ala Leu GIu Leu lie Lys Lys GIy Tyr 1040 1045 1050

Thr GIn GIn Leu Ala Phe Arg GIn Pro Ser Ser Ala Phe Ala Ala 1055 1060 1065

Phe VaI Lys Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr VaI VaI 1070 1075 1080

Lys VaI Phe Ser Leu Ala VaI Asn Leu lie Ala lie Asp Ser GIn 1085 1090 1095

VaI Leu Cys GIy Ala VaI Lys Trp Leu lie Leu GIu Lys GIn Lys 1100 1105 1110

Pro Asp GIy VaI Phe GIn GIu Asp Ala Pro VaI lie His GIn GIu 1115 1120 1125

Met lie GIy GIy Leu Arg Asn Asn Asn GIu Lys Asp Met Ala Leu 1130 1135 1140

Thr Ala Phe VaI Leu lie Ser Leu GIn GIu Ala Lys Asp lie Cys 1145 1150 1155

GIu GIu GIn VaI Asn Ser Leu Pro GIy Ser lie Thr Lys Ala GIy 1160 1165 1170

Asp Phe Leu GIu Ala Asn Tyr Met Asn Leu Gin Arg Ser Tyr Thr 1175 1180 1185

VaI Ala lie Ala GIy Tyr Ala Leu Ala GIn Met GIy Arg Leu Lys 1190 1195 1200 GIy Pro Leu Lea Asn Lys Phe Leu Thr Thr A3 a Lys Asp Lys Asn 1205 1210 1215

Arg Trp GIu Asp Pro GIy Lys GIn Leu Tyr Asn VaI GIu Ala Thr 1220 1225 1230

Ser Tyr Ala Leu Leu Ala Leu Leu GIn Leu Lys Asp Phe Asp Phe 1235 1240 1245

VaI Pro Pro VaI VaI Arg Trp Leu Asn GIu GIn Arg Tyr Tyr GIy 1250 1255 1260

GIy GIy Tyr GIy Ser Thr GIn Ala Ser His Leu His GIy VaI Pro 1265 1270 1275

Ser Leu GIy Ser lie Pro Lys GIy Arg Pro 1280 1285

<210> 15 <211> 1744

<212> PRT

<213> Artificial sequence

<220> <223> complement component 4B

<400> 15

Met Arg Leu Leu Trp GIy Leu lie Trp Ala Ser Ser Phe Phe Thr Leu 1 5 10 15

Ser Leu GIn Lys Pro Arg Leu Leu Leu Phe Ser Pro Ser VaI VaI His 20 25 30

Leu GIy VaI Pro Leu Ser VaI GIy VaI GIn Leu GIn Asp VaI Pro Arg 35 40 45

GIy Gin VaI VaI Lys GIy Ser VaI Phe Leu Arg Asn Pro Ser Arg Asn 50 55 60

Asn VaI Pro Cys Ser Pro Lys VaI Asp Phe Thr Leu Ser Ser GIu Arg 65 70 75 80

Asp Phe Ala Leu Leu Ser Leu GIn VaI Pro Leu Lys Asp Ala Lys Ser 85 90 95

Cys GIy Leu His Gin Leu Leu Arg GIy Pro GIu VaI GIn Leu VaI Ala 100 105 110

His Ser Pro Trp Leu Lys Asp Ser Leu Ser Arg Thr Thr Asn lie GIn 115 120 125

GIy lie Asn Leu Leu Phe Ser Ser Arg Arg GIy His Leu Phe Leu GIn 130 135 140

Thr Asp GIn Pro lie Tyr Asn Pro GIy GIn Arg VaI Arg Tyr Arg VaI 145 150 155 160

Phe Ala Leu Asp GIn Lys Met Arg Pro Ser Thr Asp Thr lie Thr VaI 165 170 175

Met VaI GIu Asn Ser His GIy Leu Arg VaI Arg Lys Lys GIu VaI Tyr 180 185 190

Met Pro Ser Ser lie Phe GIn Asp Asp Phe VaI lie Pro Asp lie Ser 195 200 205

GIu Pro GIy Thr Trp Lys lie Ser Ala Arg Phe Ser Asp GIy Leu GIu 210 215 220

Ser Asn Ser Ser Thr GIn Phe GIu VaI Lys Lys Tyr VaI Leu Pro Asn 225 230 235 240

Phe GIu VaI Lys lie Thr Pro GIy Lys Pro Tyr lie Leu Thr VaI Pro 245 250 255 GIy His Leu Asp GIu Met GIn Leu Asp lie GIn Ala Arg Tyr lie Tyr 260 265 270

GIy Lys Pro VaI GIn GIy VaI Ala Tyr VaI Arg Phe GIy Leu Leu Asp 275 280 285

GIu Asp GIy Lys Lys Thr Phe Phe Arg GIy Leu GIu Ser GIn Thr Lys 290 295 300

Leu VaI Asn GIy GIn Ser His lie Ser Leu Ser Lys Ala GIu Phe GIn 305 310 315 320

Asp Ala Leu GIu Lys Leu Asn Met GIy lie Thr Asp Leu GIn GIy Leu 325 330 335

Arg Leu Tyr VaI Ala Ala Ala lie lie GIu Ser Pro GIy GIy GIu Met 340 345 350

GIu GIu Ala GIu Leu Thr Ser Trp Tyr Phe VaI Ser Ser Pro Phe Ser 355 360 365

Leu Asp Leu Ser Lys Thr Lys Arg His Leu VaI Pro GIy Ala Pro Phe 370 375 380

Leu Leu GIn Ala Leu VaI Arg GIu Met Ser GIy Ser Pro Ala Ser GIy 385 390 ' 395 400

lie Pro VaI Lys VaI Ser Ala Thr VaI Ser Ser Pro GIy Ser VaI Pro 405 410 415

GIu VaI GIn Asp lie GIn GIn Asn Thr Asp GIy Ser GIy GIn VaI Ser 420 425 430

lie Pro lie lie He Pro GIn Thr He Ser GIu Leu GIn Leu Ser VaI 435 440 445

Ser Ala GIy Ser Pro His Pro Ala He Ala Arg Leu Thr VaI Ala Ala 450 455 460 Pro Pro Ser GIy GIy Pro GIy Phe Leu Ser lie GIu Arg Pro Asp Ser 465 470 475 480

Arg Pro Pro Arg VaI GIy Asp Thr Leu Asn Leu Asn Leu Arg Ala VaI

485 490 495

GIy Ser GIy Ala Thr Phe Ser His Tyr Tyr Tyr Met lie Leu Ser Arg 500 505 510

GIy GIn lie VaI Phe Met Asn Arg GIu Pro Lys Arg Thr Leu Thr Ser 515 520 525

VaI Ser VaI Phe VaI Asp His His Leu Ala Pro Ser Phe Tyr Phe VaI 530 535 540

Ala Phe Tyr Tyr His GIy Asp His Pro VaI Ala Asn Ser Leu Arg VaI 545 550 555 560

Asp VaI GIn Ala GIy Ala Cys GIu GIy Lys Leu GIu Leu Ser VaI Asp

565 570 575

GIy Ala Lys GIn Tyr Arg Asn GIy GIu Ser VaI Lys Leu His Leu GIu 580 585 590

Thr Asp Ser Leu Ala Leu VaI Ala Leu GIy Ala Leu Asp Thr Ala Leu 595 600 605

Tyr Ala Ala GIy Ser Lys Ser His Lys Pro Leu Asn Met GIy Lys VaI 610 615 620

Phe GIu Ala Met Asn Ser Tyr Asp Leu GIy Cys GIy Pro GIy GIy GIy

625 630 635 640 sp Ser Ala Leu GIn VaI Phe GIn Ala Ala GIy Leu Ala Phe Ser Asp

645 650 655

GIy Asp GIn Trp Thr Leu Ser Arg Lys Arg Leu Ser Cys Pro Lys GIu 660 665 670

Lys Thr Thr Arg Lys Lys Arg Asn VaI Asn Phe GIn Lys Ala lie Asn 675 680 685

GIu Lys Leu GIy GIn Tyr Ala Ser Pro Thr Ala Lys Arg Cys Cys GIn 690 695 700

Asp GIy VaI Thr Arg Leu Pro Met Met Arg Ser Cys GIu GIn Arg Ala 705 710 715 720

Ala Arg VaI GIn GIn Pro Asp Cys Arg GIu Pro Phe Leu Ser Cys Cys 725 730 735

Gin Phe Ala GIu Ser Leu Arg Lys Lys Ser Arg Asp Lys GIy GIn Ala 740 745 750

GIy Leu GIn Arg Ala Leu GIu He Leu GIn GIu GIu Asp Leu He Asp 755 760 765

GIu Asp Asp He Pro VaI Arg Ser Phe Phe Pro GIu Asn Trp Leu Trp 770 775 780

Arg VaI GIu Thr VaI Asp Arg Phe GIn He Leu Thr Leu Trp Leu Pro 785 790 795 800

Asp Ser Leu Thr Thr Trp GIu He His GIy Leu Ser Leu Ser Lys Thr 805 810 815

Lys GIy Leu Cys VaI Ala Thr Pro VaI GIn Leu Arg VaI Phe Arg GIu 820 825 830

Phe His Leu His Leu Arg Leu Pro Met Ser VaI Arg Arg Phe GIu GIn 835 840 845

Leu GIu Leu Arg Pro VaI Leu Tyr Asn Tyr Leu Asp Lys Asn Leu Thr 850 855 860 VaI Ser VaI His VaI Ser Pro VaI GIu GIy Leu Cys Leu Ala GIy GIy 865 870 875 880

GIy GIy Leu Ala GIn GIn VaI Leu VaI Pro Ala GIy Ser Ala Arg Pro 885 890 895

VaI Ala Phe Ser VaI VaI Pro Thr Ala Ala Ala Ala VaI Ser Leu Lys 900 905 910

VaI VaI Ala Arg GIy Ser Phe GIu Phe Pro VaI GIy Asp Ala VaI Ser 915 920 925

Lys VaI Leu GIn lie GIu Lys GIu GIy Ala lie His Arg GIu GIu Leu 930 935 940

VaI Tyr GIu Leu Asn Pro Leu Asp His Arg GIy Arg Thr Leu GIu lie 945 950 955 960

Pro GIy Asn Ser Asp Pro Asn Met lie Pro Asp GIy Asp Phe Asn Ser 965 970 975

Tyr VaI Arg VaI Thr Ala Ser Asp Pro Leu Asp Thr Leu GIy Ser GIu 980 985 990

GIy Ala Leu Ser Pro GIy GIy VaI Ala Ser Leu Leu Arg Leu Pro Arg 995 1000 1005

GIy Cys GIy GIu GIn Thr Met He Tyr Leu Ala Pro Thr Leu Ala 1010 1015 1020

Ala Ser Arg Tyr Leu Asp Lys Thr GIu GIn Trp Ser Thr Leu Pro 1025 1030 1035

Pro GIu Thr Lys Asp His Ala VaI Asp Leu He GIn Lys GIy Tyr 1040 1045 1050

Met Arg He GIn GIn Phe Arg Lys Ala Asp GIy Ser Tyr Ala Ala 1055 1060 1065 Trp Leu Ser Arg Asp Ser Ser Thr Trp Leu Thr Ala Phe VaI Leu

1070 1075 1080

Lys VaI Leu Ser Leu Ala GIn GIu GIn VaI GIy GIy Ser Pro GIu

1085 1090 1095

Lys Leu GIn GIu Thr Ser Asn Trp Leu Leu Ser GIn GIn GIn Ala

1100 1105 1110

Asp GIy Ser Phe GIn Asp Leu Ser Pro VaI lie His Arg Ser Met

1115 1120 1125

GIn GIy GIy Leu VaI GIy Asn Asp GIu Thr VaI Ala Leu Thr Ala 1130 1135 1140

Phe VaI Thr lie Ala Leu His His GIy Leu Ala VaI Phe GIn Asp

1145 1150 1155

GIu GIy Ala GIu Pro Leu Lys GIn Arg VaI GIu Ala Ser lie Ser

1160 1165 1170

Lys Ala Asn Ser Phe Leu GIy GIu Lys Ala Ser Ala GIy Leu Leu

1175 1180 1185

GIy Ala His Ala Ala Ala lie Thr Ala Tyr Ala Leu Ser Leu Thr

1190 1195 1200

Lys Ala Pro VaI Asp Leu Leu GIy VaI Ala His Asn Asn Leu Met 1205 1210 1215

Ala Met Ala GIn GIu Thr GIy Asp Asn Leu Tyr Trp GIy Ser VaI

1220 1225 1230

Thr GIy Ser Gin Ser Asn Ala VaI Ser Pro Thr Pro Ala Pro Arg

1235 1240 1245

Asn Pro Ser Asp Pro Met Pro GIn Ala Pro Ala Leu Trp lie GIu 1250 1255 1260

Thr Thr Ala Tyr Ala Leu Leu His Leu Leu Leu His GIu GIy Lys . 1265 1270 1275

Ala GIu Met Ala Asp GIn Ala Ser Ala Trp Leu Thr Arg GIn GIy 1280 1285 1290

Ser Phe GIn GIy GIy Phe Arg Ser Thr GIn Asp Thr VaI lie Ala 1295 1300 1305

Leu Asp Ala Leu Ser Ala Tyr Trp lie Ala Ser His Thr Thr GIu 1310 1315 1320

GIu Arg GIy Leu Asn VaI Thr Leu Ser Ser Thr GIy Arg Asn GIy 1325 1330 1335

Phe Lys Ser His Ala Leu GIn Leu Asn Asn Arg GIn lie Arg GIy 1340 1345 1350

Leu GIu GIu GIu Leu GIn Phe Ser Leu GIy Ser Lys lie Asn VaI 1355 1360 1365

Lys VaI GIy GIy Asn Ser Lys GIy Thr Leu Lys VaI Leu Arg Thr 1370 1375 1380

Tyr Asn VaI Leu Asp Met Lys Asn Thr Thr Cys GIn Asp Leu GIn 1385 1390 1395

lie GIu VaI Thr VaI Lys GIy His VaI GIu Tyr Thr Met GIu Ala 1400 1405 1410 sn GIu Asp Tyr GIu Asp Tyr GIu Tyr Asp GIu Leu Pro Ala Lys 1415 1420 1425 sp Asp Pro Asp Ala Pro Leu GIn Pro VaI Thr Pro Leu Gin Leu 1430 1435 1440 Phe GIu GIy Arg Arg Λsn Arg Arg Arg Arg GIu Ala Pro Lys VaI

1445 1450 1455

VaI GIu GIu GIn GIu Ser Arg VaI His Tyr Thr VaI Cys lie Trp 1460 1465 1470

Arg Asn GIy Lys VaI GIy Leu Ser GIy Met Ala lie Ala Asp VaI

1475 1480 1485

Thr Leu Leu Ser GIy Phe His Ala Leu Arg Ala Asp Leu GIu Lys

1490 1495 1500

Leu Thr Ser Leu Ser Asp Arg Tyr VaI Ser His Phe GIu Thr GIu

1505 1510 1515

GIy Pro His VaI Leu Leu Tyr Phe Asp Ser VaI Pro Thr Ser Arg

1520 1525 1530

GIu Cys VaI GIy Phe GIu Ala VaI GIn GIu VaI Pro VaI GIy Leu 1535 1540 1545

VaI GIn Pro Ala Ser Ala Thr Leu Tyr Asp Tyr Tyr Asn Pro GIu

1550 1555 1560

Arg Arg Cys Ser VaI Phe Tyr GIy Ala Pro Ser Lys Ser Arg Leu

1565 1570 1575

Leu Ala Thr Leu Cys Ser Ala GIu VaI Cys GIn Cys Ala GIu GIy

1580 1585 1590

Lys Cys Pro Arg GIn Arg Arg Ala Leu GIu Arg GIy Leu GIn Asp

1595 1600 1605

GIu Asp GIy Tyr Arg Met Lys Phe Ala Cys Tyr Tyr Pro Arg VaI 1610 1615 1620

GIu Tyr GIy Phe GIn VaI Lys VaI Leu Arg GIu Asp Ser Arg Ala

1625 1630 1635 Ala Phe Arg Leu Phe GIu Thr Lys lie Thr GIn VaI Leu His Phe 1640 1645 1650

Thr Lys Asp VaI Lys Ala Ala Ala Asn GIn Met Arg Asn Phe Leu 1655 1660 1665

VaI Arg Ala Ser Cys Arg Leu Arg Leu GIu Pro GIy Lys GIu Tyr 1670 1675 1680

Leu lie Met GIy Leu Asp GIy Ala Thr Tyr Asp Leu GIu GIy His 1685 1690 1695

Pro GIn Tyr Leu Leu Asp Ser Asn Ser Trp lie GIu GIu Met Pro 1700 1705 1710

Ser GIu Arg Leu Cys Arg Ser Thr Arg GIn Arg Ala Ala Cys Ala 1715 1720 1725

GIn Leu Asn Asp Phe Leu GIn GIu Tyr GIy Thr GIn GIy Cys GIn 1730 1735 1740

VaI

<210> 16

<211> 3674

<212> PRT

<213> Artificial sequence

<220>

<223> beta V spectrin

<400> 16

Met Ala GIy GIn Pro His Ser Pro Arg GIu Leu Leu GIy Ala Ala GIy 1 5 10 15 His Arg Ser Arg Arg Pro Ser Thr GIu Leu Arg VaI Pro Pro Ser Pro 20 25 30

Ser Leu Thr Met Asp Ser GIn Tyr GIu Thr GIy His lie Arg Lys Leu 35 40 45

GIn Ala Arg His Met GIn Met GIn GIu Lys Thr Phe Thr Lys Trp lie 50 55 60

Asn Asn VaI Phe GIn Cys GIy GIn Ala GIy lie Lys lie Arg Asn Leu 65 70 75 80

Tyr Thr GIu Leu Ala Asp GIy lie His Leu Leu Arg Leu Leu GIu Leu 85 90 95

He Ser GIy GIu Ala Leu Pro Pro Pro Ser Arg GIy Arg Leu Arg VaI 100 105 110

His Phe Leu GIu Asn Ser Ser Arg Ala Leu Ala Phe Leu Arg Ala Lys 115 120 125

VaI Pro VaI Pro Leu He GIy Pro GIu Asn He VaI Asp GIy Asp GIn 130 135 140

Thr Leu He Leu GIy Leu He Trp VaI He He Leu Arg Phe GIn He 145 150 155 160

Ser His He Ser Leu Asp Lys GIu GIu Phe GIy Ala Ser Ala Ala Leu 165 170 175

Leu Ser Thr Lys GIu Ala Leu Leu VaI Trp Cys GIn Arg Lys Thr Ala 180 185 190

Ser Tyr Thr Asn VaI Asn He Thr Asp Phe Ser Arg Ser Trp Ser Asp 195 200 205

GIy Leu GIy Phe Asn Ala Leu He His Ala His Arg Pro Asp Leu Leu 210 215 220 Asp Tyr GIy Ser Leu Arg Pro Asp Arg Pro Leu His Asn Leu Ala Phe

225 230 235 240.

Ala Phe Leu VaI Ala GIu GIn GIu Leu GIy lie Ala GIn Leu Leu Asp

245 250 255

Pro GIu Asp VaI Ala Ala Ala GIn Pro Asp GIu Arg Ser He Met Thr

260 265 270

Tyr VaI Ser Leu Tyr Tyr His Tyr Cys Ser Arg Leu His GIn GIy GIn 275 280 285

Thr VaI GIn Arg Arg Leu Thr Lys He Leu Leu GIn Leu GIn GIu Thr 290 295 300

GIu Leu Leu GIn Thr GIn Tyr GIu GIn Leu VaI Ala Asp Leu Leu Arg 305 310 315 320

Trp He Ala GIu Lys GIn Met GIn Leu GIu Ala Arg Asp Phe Pro Asp

325 330 335

Ser Leu Pro Ala Met Arg GIn Leu Leu Ala Ala Phe Thr He Phe Arg 340 345 350

Thr GIn GIu Lys Pro Pro Arg Leu GIn GIn Arg GIy Ala Ala GIu Ala 355 360 365

Leu Leu Phe Arg Leu GIn Thr Ala Leu GIn Ala GIn Asn Arg Arg Pro 370 375 380

Phe Leu Pro His GIu GIy Leu GIy Leu Ala GIu Leu Ser GIn Cys Trp 385 390 395 400

Ala GIy Leu GIu Trp Ala GIu Ala Ala Arg Ser GIn Ala Leu GIn GIn

405 410 415

Arg Leu Leu GIn Leu GIn Arg Leu GIu Thr Leu Ala Arg Arg Phe GIn 420 425 430

His Lys Ala Ala Leu Arg GIu Ser Phe Leu Lys Asp Ala GIu GIn VaI 435 440 445

Leu Asp GIn Ala Arg Ala Pro Pro Ala Ser Leu Ala Thr VaI GIu Ala 450 455 460

Ala VaI GIn Arg Leu GIy Met Leu GIu Ala GIy lie Leu Pro GIn GIu 465 470 475 480

GIy Arg Phe GIn Ala Leu Ala GIu lie Ala Asp lie Leu Arg GIn GIu 485 490 495

GIn Tyr His Ser Trp Ala Asp VaI Ala Arg Arg GIn GIu GIu VaI Thr 500 505 510

VaI Arg Trp GIn Arg Leu Leu GIn His Leu GIn GIy GIn Arg Lys GIn 515 520 525

VaI Ala Asp Met GIn Ala VaI Leu Ser Leu Leu GIn GIu VaI GIu Ala 530 535 540

Ala Ser His GIn Leu GIu GIu Leu GIn GIu Pro Ala Arg Ser Thr Ala 545 550 555 560

Cys GIy GIn GIn Leu Ala GIu VaI VaI GIu Leu Leu GIn Arg His Asp 565 570 575

Leu Leu GIu Ala GIn VaI Ser Ala His GIy Ala His VaI Ser His Leu 580 585 590

Ala GIn GIn Thr Ala GIu Leu Asp Ser Ser Leu GIy Thr Ser VaI GIu 595 600 605

VaI Leu GIn Ala Lys Ala Arg Thr Leu Ala GIn Leu GIn GIn Ser Leu 610 615 620 VaI Ala Leu VaI Arg Ala Arg Arg Ala Leu Leu GIu GIn Thr Leu GIn 625 630 635 640

Arg Ala GIu Phe Leu Arg Asn Cys GIu GIu GIu GIu Ala Trp Leu Lys 645 650 655

GIu Cys GIy GIn Arg VaI GIy Asn Ala Ala Leu GIy Arg Asp Leu Ser 660 665 670

GIn lie Ala GIy Ala Leu GIn Lys His Lys Ala Leu GIu Ala GIu VaI 675 680 685

His Arg His GIn Ala VaI Cys VaI Asp Leu VaI Arg Arg GIy Arg Asp 690 695 700

Leu Ser Ala Arg Arg Pro Pro Thr GIn Pro Asp Pro GIy GIu Arg Ala 705 710 715 720

GIu Ala VaI GIn GIy GIy Trp GIn Leu Leu GIn Thr Arg VaI VaI GIy 725 730 735

Arg GIy Ala Arg Leu GIn Thr Ala Leu Leu VaI Leu GIn Tyr Phe Ala 740 745 750

Asp Ala Ala GIu Ala Ala Ser Trp Leu Arg GIu Arg Arg Ser Ser Leu 755 760 765

GIu Arg Ala Ser Cys GIy GIn Asp GIn Ala Ala Ala GIu Thr Leu Leu 770 775 780

Arg Arg His VaI Arg Leu GIu Arg VaI Leu Arg Ala Phe Ala Ala GIu 785 790 795 800

Leu Arg Arg Leu GIu GIu GIn GIy Arg Ala Ala Ser Ala Arg Ala Ser 805 810 815

Leu Phe Thr VaI Asn Ser Ala Leu Ser Pro Pro GIy GIu Ser Leu Arg 820 825 830 Asn Pro GIy Pro Trp Ser GIu Ala Ser Cys His Pro GIy Pro GIy Asp 835 840 845

Ala Trp Lys Met Ala Leu Pro Ala GIu Pro Asp Pro Asp Phe Asp Pro 850 855' 860

Asn Thr lie Leu GIn Thr GIn Asp His Leu Ser GIn Asp Tyr GIu Ser 865 870 875 880

Leu Arg Ala Leu Ala GIn Leu Arg Arg Ala Arg Leu GIu GIu Ala Met 885 890 895

Ala Leu Phe GIy Phe Cys Ser Ser Cys GIy GIu Leu GIn Leu Trp Leu 900 905 ^• 910

GIu Lys GIn Thr VaI Leu Leu GIn Arg VaI GIn Pro GIn Ala Asp Thr 915 920 925

Leu GIu VaI Met GIn Leu Lys Tyr GIu Asn Phe Leu Thr Ala Leu Ala 930 935 940

VaI GIy Lys GIy Leu Trp Ala GIu VaI Ser Ser Ser Ala GIu GIn Leu 945 950 955 960

Arg Gin Arg Tyr Pro GIy Asn Ser Thr GIn lie GIn Arg GIn GIn GIu 965 970 975

GIu Leu Ser GIn Arg Trp GIy GIn Leu GIu Ala Leu Lys Arg GIu Lys 980 985 990

Ala VaI GIn Leu Ala His Ser VaI GIu VaI Cys Ser Phe Leu GIn GIu 995 1000 1005

Cys GIy Pro Thr GIn VaI GIn Leu Arg Asp VaI Leu Leu GIn Leu 1010 1015 1020

GIu Ala Leu GIn Pro GIy Ser Ser GIu Asp Thr Cys His Ala Leu 1025 1030 1035

GIn Leu Ala GIn Lys Lys Thr Leu VaI Leu GIu Arg Arg VaI Hxs 1040 1045 1050

Phe Leu GIn Ser VaI VaI VaI Lys VaI GIu GIu Pro GIy Tyr Ala 1055 1060 1065

GIu Ser GIn Pro Leu GIn GIy GIn VaI GIu Thr Leu GIn GIy Leu 1070 1075 1080

Leu Lys GIn VaI GIn GIu GIn VaI Ala GIn Arg Ala Arg Arg GIn 1085 1090 1095

Ala GIu Thr GIn Ala Arg GIn Ser Phe Leu GIn GIu Ser GIn GIn 1100 1105 1110

Leu Leu Leu Trp Ala GIu Ser VaI GIn Ala GIn Leu Arg Ser Lys 1115 1120 1125

GIu VaI Ser VaI Asp VaI Ala Ser Ala GIn Arg Leu Leu Arg GIu 1130 1135 1140

His GIn Asp Leu Leu GIu GIu lie His Leu Trp GIn GIu Arg Leu 1145 1150 1155

GIn GIn Leu Asp Ala GIn Ser GIn Pro Met Ala Ala Leu Asp Cys 1160 1165 1170

Pro Asp Ser GIn GIu VaI Pro Asn Thr Leu Arg VaI Leu GIy GIn 1175 1180 1185

GIn GIy GIn GIu Leu Lys VaI Leu Trp GIu GIn Arg GIn GIn Trp 1190 1195 1200

Leu GIn GIu GIy Leu GIu Leu Gin Lys Phe GIy Arg GIu VaI Asp 1205 1210 1215 GIy Phε Thr Ala Thr Cys Ala Asn His GIn Ala Trp Leu His Leu

1220 1225 1230

Asp Asn Leu GIy GIu Asp VaI Arg GIu Ala Leu Ser Leu Leu GIn 1235 1240 1245

GIn His Arg GIu Phe GIy Arg Leu Leu Ser Thr Leu GIy Pro Arg

1250 1255 1260

Ala GIu Ala Leu Arg Ala His GIy GIu Lys Leu VaI GIn Ser GIn

1265 1270 1275

His Pro Ala Ala His Thr VaI Arg GIu GIn Leu GIn Ser He GIn

1280 1285 1290

Ala GIn Trp Thr Arg Leu GIn GIy Arg Ser GIu GIn Arg Arg Arg

1295 1300 1305

GIn Leu Leu Ala Ser Leu GIn Leu GIn GIu Trp Lys GIn Asp VaI 1310 1315 1320

Ala GIu Leu Met GIn Trp Met GIu GIu Lys GIy Leu Met Ala Ala

1325 1330 1335

His GIu Pro Ser GIy Ala Arg Arg Asn lie Leu GIn Thr Leu Lys

1340 1345 1350

Arg His GIu Ala Ala GIu Ser GIu Leu Leu Ala Thr Arg Arg His

1355 1360 1365

VaI GIu Ala Leu GIn GIn VaI GIy Arg GIu Leu Leu Ser Arg Arg

1370 1375 1380

Pro Cys GIy GIn GIu Asp He GIn Thr Arg Leu GIn GIy Leu Arg 1385 1390 1395

Ser Lys Trp GIu Ala Leu Asn Arg Lys Met Thr GIu Arg GIy Asp

1400 1405 1410 GIu Leu GIn GIn Ala GIy GIn GIn GIu GIn Leu Leu Arg GIn Leu

1415 1420 1425

GIn Asp Ala Lys GIu GIn Leu GIu GIn Leu GIu GIy Ala Leu GIn

1430 1435 1440

Ser Ser GIu Thr GIy GIn Asp Leu Arg Ser Ser GIn Arg Leu GIn

1445 1450 1455

Lys Arg His GIn GIn Leu GIu Ser GIu Ser Arg Thr Leu Ala Ala

1460 1465 1470

Lys Met Ala Ala Leu Ala Ser Met Ala His GIy Met Ala Ala Ser 1475 1480 1485

Pro Ala lie Leu GIu GIu Thr GIn Lys His Leu Arg Arg Leu GIu

1490 1495 1500

Leu Leu GIn GIy His Leu Ala lie Arg GIy Leu GIn Leu GIn Ala

1505 1510 1515

Ser VaI GIu Leu His GIn Phe Cys His Leu Ser Asn Met GIu Leu

1520 1525 1530

Ser Trp VaI Ala GIu His Met Pro His GIy Ser Pro Thr Ser Tyr

1535 1540 1545

Thr GIu Cys Leu Asn GIy Ala GIn Ser Leu His Arg Lys His Lys 1550 1555 1560

GIu Leu GIn VaI GIu VaI Lys Ala His GIn GIy GIn VaI GIn Arg

1565 1570 1575

VaI Leu Ser Ser GIy Arg Ser Leu Ala Ala Ser GIy His Pro GIn

1580 1585 1590

Ala GIn His lie VaI GIu GIn Cys GIn GIu Leu GIu GIy His Trp 1595 1600 1605

Ala GIu Leu GIu Arg Ala Cys GIu Ala Arg Ala GIn Cys Leu GIn 1610 1615 1620

Gin Ala VaI Thr Phe GIn GIn Tyr Phe Leu Asp VaI Ser GIu Leu 1625 1630 1635

GIu GIy Trp VaI GIu GIu Lys Arg Pro Leu VaI Ser Ser Arg Asp 1640 1645 1650

Tyr GIy Arg Asp GIu Ala Ala Thr Leu Arg Leu lie Asn Lys His 1655 1660 1665

GIn Ala Leu GIn GIu GIu Leu Ala lie Tyr Trp Ser Ser Met GIu 1670 1675 1680

GIu Leu Asp GIn Thr Ala GIn Thr Leu Thr GIy Pro GIu VaI Pro 1685 1690 1695

GIu GIn GIn Arg VaI VaI GIn GIu Arg Leu Arg GIu GIn Leu Arg 1700 1705 1710

Ala Leu GIn GIu Leu Ala Ala Thr Arg Asp Arg GIu Leu GIu GIy 1715 1720 1725

Thr Leu Arg Leu His GIu Phe Leu Arg GIu Ala GIu Asp Leu GIn 1730 1735 1740

GIy Trp Leu Ala Ser GIn Lys GIn Ala Ala Lys GIy GIy GIu Ser 1745 1750 1755

Leu GIy GIu Asp Pro GIu His Ala Leu His Leu Cys Thr Lys Phe 1760 1765 1770

Ala Lys Phe GIn His GIn VaI GIu Met GIy Ser GIn Arg VaI Ala 1775 1780 1785 Ala Cys Arg Leu Leu Ala GIu Ser Leu Leu. GIu Arg GIy His Ser 1790 1795 1800

Ala GIy Pro Met VaI Arg GIn Arg GIn GIn Asp Leu GIn Thr Ala 1805 1810 1815

Trp Ser GIu Leu Trp GIu Leu Thr GIn Ala Arg GIy His Ala Leu 1820 1825 1830

Arg Asp Thr GIu Thr Thr Leu Arg VaI His Arg Asp Leu Leu GIu 1835 1840 1845

VaI Leu Thr Gin VaI GIn GIu Lys Ala Thr Ser Leu Pro Asn Asn 1850 1855 1860

VaI Ala Arg Asp Leu Cys GIy Leu GIu Ala GIn Leu Arg Ser His 1865 1870 1875

GIn GIy Leu GIu Arg GIu Leu VaI GIy Thr GIu Arg GIn Leu GIn 1880 1885 1890

GIu Leu Leu GIu Thr Ala GIy Arg VaI GIn Lys Leu Cys Pro GIy 1895 1900 1905

Pro GIn Ala His Ala VaI GIn GIn Arg GIn GIn Ala VaI Thr GIn 1910 1915 1920

Ala Trp Ala VaI Leu GIn Arg Arg Met GIu GIn Arg Arg Ala GIn 1925 1930 1935

Leu GIu Arg Ala Arg Leu Leu Ala Arg Phe Arg Thr Ala VaI Arg 1940 1945 1950

Asp Tyr Ala Ser Trp Ala Ala Arg VaI Arg GIn Asp Leu GIn VaI 1955 1960 1965

GIu GIu Ser Ser GIn GIu Pro Ser Ser GIy Pro Leu Lys Leu Ser 1970 1975 1980 Ala His GIn Trp Leu Arg Ala GIu Leu GIu Ala Arg GIu Lys Leu 1985 1990 1995

Trp GIn GIn Ala Thr GIn Leu GIy GIn GIn Ala Leu Leu Ala Ala 2000 2005 2010

GIy Thr Pro Thr Lys GIu VaI Gin GIu GIu Leu Arg Ala Leu GIn 2015 2020 2025

Asp GIn Arg Asp GIn VaI Tyr GIn Thr Trp Ala Arg Lys GIn GIu 2030 2035 2040

Arg Leu GIn Ala GIu GIn GIn GIu GIn Leu Phe Leu Arg GIu Cys 2045 2050 2055

GIy Arg Leu GIu GIu lie Leu Ala Ala GIn GIu VaI Ser Leu Lys 2060 2065 2070

Thr Ser Ala Leu GIy Ser Ser VaI GIu GIu VaI GIu GIn Leu He 2075 2080 2085

Arg Lys His GIu VaI Phe Leu Lys VaI Leu Thr Ala GIn Asp Lys 2090 2095 2100

Lys GIu Ala Ala Leu Arg GIu Arg Leu Lys Thr Leu Arg Arg Pro 2105 2110 2115

Arg VaI Arg Asp Arg Leu Pro lie Leu Leu GIn Arg Arg Met Arg 2120 2125 2130

VaI Lys GIu Leu Ala GIu Ser Arg GIy His Ala Leu His Ala Ser 2135 2140 2145

Leu Leu Met Ala Ser Phe Thr GIn Ala Ala Thr GIn Ala GIu Asp 2150 2155 2160

Trp He GIn Ala Trp Ala GIn GIn Leu Lys GIu Pro VaI Pro Pro 2165 2170 2175

GIy Asp Leu Arg Asp Lys Leu Lys Pro Leu Leu Lys His GIn Ala

2180 2185 2190

Phe GIu Ala GIu VaI GIn Ala His GIu GIu VaI Met Thr Ser VaI

2195 2200 2205

Ala Lys Lys GIy GIu Ala Leu Leu Ala GIn Ser His Pro Arg Ala 2210 2215 2220

GIy GIu VaI Ser GIn Arg Leu GIn GIy Leu Arg Lys His Trp GIu

2225 2230 2235

Asp Leu Arg GIn Ala Met Ala Leu Arg GIy GIn GIu Leu GIu Asp

2240 2245 2250

Arg Arg Asn Phe Leu GIu Phe Leu GIn Arg VaI Asp Leu Ala GIu

2255 2260 2265

Ala Trp lie GIn GIu Lys GIu VaI Lys Met Asn VaI GIy Asp Leu

2270 2275 2280

GIy GIn Asp Leu GIu His Cys Leu GIn Leu Arg Arg Arg Leu Arg 2285 2290 2295

GIu Phe Arg GIy Asn Ser Ala GIy Asp Thr VaI GIy Asp Ala Cys

2300 2305 2310

lie Arg Ser lie Ser Asp Leu Ser Leu GIn Leu Lys Asn Arg Asp

2315 2320 2325

Pro GIu GIu VaI Lys lie lie Cys GIn Arg Arg Ser GIn Leu Asn

2330 2335 2340 sn Arg Trp Ala Ser Phe His GIy Asn Leu Leu Arg Tyr GIn GIn

2345 2350 2355 GIn Leu GIu GIy Ala Leu GIu He His VaI Leu Ser Arg GIu Leu

2360 2365 2370

Asp Asn VaI Thr Lys Arg He GIn GIu Lys GIu Ala Leu He GIn 2375 2380 2385

Ala Leu Asp Cys GIy Lys Asp Leu GIu Ser VaI GIn Arg Leu Leu

2390 2395 2400

Arg Lys His GIu GIu Leu GIu Arg GIu VaI His Pro He GIn Ala

2405 2410 2415

GIn VaI GIu Ser Leu GIu Arg GIu VaI GIy Arg Leu Cys GIn Arg

2420 2425 2430

Ser Pro GIu Ala Ala His GIy Leu Arg His Arg GIn GIn GIu VaI

2435 2440 2445

Ala GIu Ser Trp Trp GIn Leu Arg Ser Arg Ala GIn Lys Arg Arg 2450 2455 2460

GIu Ala Leu Asp Ala Leu His GIn Ala GIn Lys Leu GIn Ala Met

2465 2470 2475

Leu GIn GIu Leu Leu VaI Ser Ala GIn Arg Leu Arg Ala GIn Met

2480 2485 2490

Asp Thr Ser Pro Ala Pro Arg Ser Pro VaI GIu Ala Arg Arg Met

2495 2500 2505

Leu GIu GIu His GIn GIu Cys Lys Ala GIu Leu Asp Ser Trp Thr

2510 2515 2520

Asp Ser He Ser Leu Ala Arg Ser Thr GIy GIn GIn Leu Leu Thr 2525 2530 2535

Ala GIy His Pro Phe Ser Ser Asp He Arg GIn VaI Leu Ala GIy

2540 2545 2550 Leu GIu GIn GIu Leu Ser Ser Leu GIu GIy Ala Trp GIn GIu His

2555 2560 2565

GIn Leu GIn Leu GIn GIn Ala Leu GIu Leu GIn Leu Phe Leu Ser

2570 2575 2580

Ser VaI GIu Lys Met GIu Arg Trp Leu Cys Ser Lys GIu Asp Ser

2585 2590 2595

Leu Ala Ser GIu GIy Leu Trp Asp Pro Leu Ala Pro Met GIu Pro

2600 2605 2610

Leu Leu Trp Lys His Lys Met Leu GIu Trp Asp Leu GIu VaI GIn 2615 2620 2625

Ala GIy Lys lie Ser Ala Leu GIu Ala Thr Ala Arg GIy Leu His

2630 2635 2640

GIn GIy GIy His Pro GIu Ala GIn Ser Ala Leu GIy Arg Cys GIn

2645 2650 2655

Ala Met Leu Leu Arg Lys GIu Ala Leu Phe Arg GIn Ala GIy Thr

2660 2665 2670

Arg Arg His Arg Leu GIu GIu Leu Arg GIn Leu GIn Ala Phe Leu

2675 2680 2685

GIn Asp Ser GIn GIu VaI Ala Ala Trp Leu Arg GIu Lys Asn Leu 2690 2695 2700

VaI Ala Leu GIu GIu GIy Leu Leu Asp Thr Ala Met Leu Pro Ala

2705 2710 2715

GIn Leu GIn Lys GIn GIn Asn Phe GIn Ala GIu Leu Asp Ala Ser

2720 2725 2730

Met His GIn GIn GIn GIu Leu GIn Arg GIu GIy GIn Arg Leu Leu 2735 2740 2745

GIn GIy GIy His Pro Ala Ser GIu Ala lie GIn GIu Arg Leu GIu 2750 2755 2760

GIu Leu GIy Ala Leu Trp GIy GIu Leu GIn Asp Asn Ser GIn Lys 2765 2770 2775

Lys VaI Ala Lys Leu GIn Lys Ala Cys GIu Ala Leu Arg Leu Arg 2780 2785 2790

Arg Ser Met GIu GIu Leu GIu Asn Trp Leu GIu Pro lie GIu VaI 2795 2800 2805

GIu Leu Arg Ala Pro Thr VaI GIy GIn Ala Leu Pro GIy VaI GIy 2810 2815 2820

GIu Leu Leu GIy Thr GIn Arg GIu Leu GIu Ala Ala VaI Asp Lys 2825 2830 2835

Lys Ala Arg GIn Ala GIu Ala Leu Leu GIy GIn Ala GIn Ala Phe 2840 2845 2850

VaI Arg GIu GIy His Cys Leu Ala GIn Asp VaI GIu GIu GIn Ala 2855 2860 2865

Arg Arg Leu Leu GIn Arg Phe Lys Ser Leu Arg GIu Pro Leu GIn 2870 2875 2880

GIu Arg Arg Thr Ala Leu GIu Ala Arg Ser Leu Leu Leu Lys Phe 2885 2890 2895

Phe Arg Asp Ala Asp GIu GIu Met Ala Trp VaI GIn GIu Lys Leu 2900 2905 2910

Pro Leu Ala Ala Ala GIn Asp Tyr GIy GIn Ser Leu Ser Ala VaI 2915 2920 2925 Arg His Leu GIn GIu GIn His GIn Asn Leu GIu Ser GIu Met Ser 2930 2935 2940

Ser His GIu Ala Leu Thr Arg VaI VaI Leu GIy Thr GIy Tyr Lys 2945 2950 . 2955

Leu VaI GIn Ala GIy His Phe Ala Ala His GIu VaI Ala Ala Arg 2960 2965 2970

VaI GIn GIn Leu GIu Lys Ala Met Ala His Leu Arg Ala GIu Ala 2975 2980 2985

Ala Arg Arg Arg Leu Leu Leu GIn GIn Ala GIn GIu Ala GIn GIn 2990 2995 3000

Phe Leu Thr GIu Leu Leu GIu Ala GIy Ser Trp Leu Ala GIu Arg 3005 3010 3015

GIy His VaI Leu Asp Ser GIu Asp Met GIy His Ser Ala GIu Ala 3020 3025 3030

Thr GIn Ala Leu Leu Arg Arg Leu GIu Ala Thr Lys Arg Asp Leu 3035 3040 3045

GIu Ala Phe Ser Pro Arg He GIu Arg Leu GIn GIn Thr Ala Ala 3050 3055 3060

Leu Leu GIu Ser Arg Lys Asn Pro GIu Ser Pro Lys VaI Leu Ala 3065 3070 3075

Gin Leu GIn Ala VaI Arg GIu Ala His Ala GIu -Leu Leu Arg Arg 3080 3085 3090

Ala GIu Ala Arg GIy His GIy Leu GIn GIu GIn Leu GIn Leu His 3095 3100 3105

GIn Leu GIu Arg GIu Thr Leu Leu Leu Asp Ala Trp Leu Thr Thr 3110 3115 3120 Lys Ala Ala Thr Ala GIu Ser GIn Asp Tyr GIy GIn Asp Leu GIu 3125 3130 3135

GIy VaI Lys VaI Leu GIu GIu Lys Phe Asp Ala Phe Arg Lys GIu 3140 3145 3150

VaI GIn Ser Leu GIy GIn Ala Lys VaI Tyr Ala Leu Arg Lys Leu 3155 3160 3165

Ala GIy Thr Leu GIu Arg GIy Ala Pro Arg Arg Tyr Pro His lie 3170 3175 3180

GIn Ala GIn Arg Ser Arg lie GIu Ala Ala Trp GIu Arg Leu Asp 3185 3190 3195

GIn Ala lie Lys Ala Arg Thr GIu Asn Leu Ala Ala Ala His GIu 3200 3205 3210

VaI His Ser Phe GIn GIn Ala Ala Ala GIu Leu GIn GIy Arg Met 3215 3220 3225

GIn GIu Lys Thr Ala Leu Met Lys GIy GIu Asp GIy GIy His Ser 3230 3235 3240

Leu Ser Ser VaI Arg Thr Leu GIn GIn GIn His Arg Arg Leu GIu 3245 3250 3255

Arg GIu Leu GIu Ala Met GIu Lys GIu VaI Ala Arg Leu GIn Thr 3260 3265 3270

GIu Ala Cys Arg Leu GIy GIn Leu His Pro Ala Ala Pro GIy GIy 3275 3280 3285

Leu Ala Lys VaI GIn GIu Ala Trp Ala Thr Leu GIn Ala Lys Ala 3290 3295 3300

GIn GIu Arg GIy Gin Trp Leu Ala GIn Ala Ala GIn GIy His Ala 3305 3310 3315

Phe Leu GIy Arg Cys GIn GIu Leu Leu Ala Trp Ala GIn GIu Arg

3320 3325 3330

Gin GIu Leu Ala Ser Ser GIu GIu Leu Ala GIu Asp VaI Ala GIy

3335 3340 3345

Ala GIu GIn Leu Leu GIy GIn His GIu GIu Leu GIy GIn GIu lie 3350 3355 3360

Arg GIu Cys Arg Leu GIn Ala GIn Asp Leu Arg GIn GIu GIy GIn

3365 3370 3375

GIn Leu VaI Asp Asn Ser His Phe Met Ser Ala GIu VaI Thr GIu

3380 3385 3390

Cys Leu GIn GIu Leu GIu GIy Arg Leu GIn GIu Leu GIu GIu Ala

3395 3400 3405

Trp Ala Leu Arg Trp GIn Arg Cys Ala GIu Ser Trp GIy Leu GIn

3410 3415 3420

Lys Leu Arg GIn Arg Leu GIu GIn Ala GIu Ala Trp Leu Ala Cys 3425 3430 3435

Trp GIu GIy Leu Leu Leu Lys Pro Asp Tyr GIy His Ser VaI Ser

3440 3445 3450

Asp VaI GIu Leu Leu Leu His Arg His GIn Asp Leu GIu Lys Leu

3455 3460 3465

Leu Ala Ala GIn GIu GIu Lys Phe Ala GIn Met GIn Lys Thr GIu

3470 3475 3480

Met GIu GIn GIu Leu Leu Leu GIn Pro GIn GIu Leu Lys Pro GIy

3485 3490 3495 Arg Ala GIy Ser Ser Leu Thr Ser Phe GIn Trp Arg Pxo Ser GIy 3500 3505 3510

His GIn GIy Leu GIy Ala GIn Leu Ala GIu Thr Arg Asp Pro GIn 3515 3520 3525

Asp Ala Lys GIy Thr Pro Thr Met GIu GIy Ser Leu GIu Phe Lys 3530 3535 3540

GIn His Leu Leu Pro GIy GIy Arg GIn Pro Ser Ser Ser Ser Trp 3545 3550 3555

Asp Ser Cys Arg GIy Asn Leu GIn GIy Ser Ser Leu Ser Leu Phe 3560 3565 3570

Leu Asp GIu Arg Met Ala Ala GIu Lys VaI Ala Ser lie Ala Leu 3575 3580 3585

Leu Asp Leu Thr GIy Ala Arg Cys GIu Arg Leu Arg GIy Arg His 3590 3595 3600

GIy Arg Lys His Thr Phe Ser Leu Arg Leu Thr Ser GIy Ala GIu 3605 3610 3615

lie Leu Phe Ala Ala Pro Ser GIu GIu GIn Ala GIu Ser Trp Trp 3620 3625 3630

Arg Ala Leu GIy Ser Thr Ala Ala GIn Ser Leu Ser Pro Lys Leu 3635 3640 3645

Lys Ala Lys Pro VaI Ser Ser Leu Asn GIu Cys Thr Thr Lys Asp 3650 3655 3660

Ala Arg Pro GIy Cys Leu Leu Arg Ser Asp Pro 3665 3670

<210> 17 <211> 314 <212> PRT

<213> Artificial sequence

<220> <223> osteopontin

<400> 17

Met Arg lie Ala VaI lie Cys Phe Cys Leu Leu GIy lie Thr Cys Ala 1 '5 10 15

lie Pro VaI Lys GIn Ala Asp Ser GIy Ser Ser GIu GIu Lys GIn Leu 20 25 30

Tyr Asn Lys Tyr Pro Asp Ala VaI Ala Thr Trp Leu Asn Pro Asp Pro 35 40 45

Ser GIn Lys GIn Asn Leu Leu Ala Pro GIn Asn Ala VaI Ser Ser GIu 50 55 60

GIu Thr Asn Asp Phe Lys GIn GIu Thr Leu Pro Ser Lys Ser Asn GIu 65 70 75 80

Ser His Asp His Met Asp Asp Met Asp Asp GIu Asp Asp Asp Asp His 85 90 95

VaI Asp Ser GIn Asp Ser lie Asp Ser Asn Asp Ser Asp Asp VaI Asp 100 105 110

Asp Thr Asp Asp Ser His GIn Ser Asp GIu Ser His His Ser Asp GIu 115 120 125

Ser Asp GIu Leu VaI Thr Asp Phe Pro Thr Asp Leu Pro Ala Thr GIu 130 135 140

VaI Phe Thr Pro VaI VaI Pro Thr VaI Asp Thr Tyr Asp GIy Arg GIy 145 150 155 160 Asp Ser VaI VaI Tyr GIy Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg ^' 165 170 175

Pro Asp lie GIn Tyr Pro Asp Ala Thr Asp GIu Asp lie Thr Ser His 180 185 190

Met GIu Ser GIu GIu Leu Asn GIy Ala Tyr Lys Ala lie Pro VaI Ala 195 200 205

GIn Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg GIy Lys Asp Ser 210 215 220

Tyr GIu Thr Ser GIn Leu Asp Asp Gin Ser Ala GIu Thr His Ser His 225 230 235 240

Lys GIn Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp GIu Ser Asn GIu 245 250 255

His Ser Asp VaI lie Asp Ser GIn GIu Leu Ser Lys VaI Ser Arg GIu 260 265 270

Phe His Ser His GIu Phe His Ser His GIu Asp Met Leu VaI VaI Asp 275 280 285

Pro Lys Ser Lys GIu GIu Asp Lys His Leu Lys Phe Arg lie Ser His 290 295 300

GIu Leu Asp Ser Ala Ser Ser GIu VaI Asn 305 310

<210> 18

<211> 189

<212> PRT

<213> Artificial sequence

<220>

<223> apolipoprotein D C400> 18

Met VaI Met Leu Leu Leu Leu Leu Ser Ala Leu Ala GIy Leu Phe GIy 1 5 10 15

Ala Ala GIu GIy GIn Ala Phe His Leu GIy Lys Cys Pro Asn Pro Pro 20 25 30

VaI GIn GIu Asn Phe Asp VaI Asn Lys Tyr Leu GIy Arg Trp Tyr GIu 35 40 45

lie GIu Lys lie Pro Thr Thr Phe GIu Asn GIy Arg Cys lie GIn Ala 50 55 60

Asn Tyr Ser Leu Met GIu Asn GIy Lys lie Lys VaI Leu Asn GIn GIu 65 70 75 80

Leu Arg Ala Asp GIy Thr VaI Asn GIn lie GIu GIy GIu Ala Thr Pro 85 90 95

VaI Asn Leu Thr GIu Pro Ala Lys Leu GIu VaI Lys Phe Ser Trp Phe 100 105 110

Met Pro Ser Ala Pro Tyr Trp lie Leu Ala Thr Asp Tyr GIu Asn Tyr 115 120 125

Ala Leu VaI Tyr Ser Cys Thr Cys lie lie GIn Leu Phe His VaI Asp 130 135 140

Phe Ala Trp lie Leu Ala Arg Asn Pro Asn Leu Pro Pro GIu Thr VaI 145 150 155 160

Asp Ser Leu Lys Asn lie Leu Thr Ser Asn Asn lie Asp VaI Lys Lys 165 170 175

Met Thr VaI Thr Asp GIn VaI Asn Cys Pro Lys Leu Ser 180 185 <210> 19

<211> 1744

<212> PRT

<213> Artificial sequence

<220>

<223> complement component 4A

<400> 19

Met Arg Leu Leu Trp GIy Leu lie Trp Ala Ser Ser Phe Phe Thr Leu 1 5 10 15

Ser Leu GIn Lys Pro Arg Leu Leu Leu Phe Ser Pro Ser VaI VaI His 20 25 30

Leu GIy VaI Pro Leu Ser VaI GIy VaI GIn Leu GIn Asp VaI Pro Arg 35 40 45

GIy GIn VaI VaI Lys GIy Ser VaI Phe Leu Arg Asn Pro Ser Arg Asn 50 55 60

Asn VaI Pro Cys Ser Pro Lys VaI Asp Phe Thr Leu Ser Ser GIu Arg 65 70 75 80

Asp Phe Ala Leu Leu Ser Leu GIn VaI Pro Leu Lys Asp Ala Lys Ser 85 90 95

Cys GIy Leu His GIn Leu Leu Arg GIy Pro GIu VaI GIn Leu VaI Ala 100 105 110

His Ser Pro Trp Leu Lys Asp Ser Leu Ser Arg Thr Thr Asn lie GIn 115 120 125

GIy lie Asn Leu Leu Phe Ser Ser Arg Arg GIy His Leu Phe Leu GIn 130 135 140

Thr Asp GIn Pro lie Tyr Asn Pro GIy GIn Arg VaI Arg Tyr Arg VaI 145 150 155 160

Phe Ala Leu Asp GIn Lys Met Arg Pro Ser Thr Asp Thr lie Thr VaI 165 170 175

Met VaI GIu Asn Ser His GIy Leu Arg VaI Arg Lys Lys GIu VaI Tyr 180 185 190

Met Pro Ser Ser lie Phe GIn Asp Asp Phe VaI lie Pro Asp He Ser 195 200 205

GIu Pro GIy Thr Trp Lys He Ser Ala Arg Phe Ser Asp GIy Leu GIu 210 215 220

Ser Asn Ser Ser Thr GIn Phe GIu VaI Lys Lys Tyr VaI Leu Pro Asn 225 230 235 240

Phe GIu VaI Lys He Thr Pro GIy Lys Pro Tyr He Leu Thr VaI Pro 245 250 255

GIy His Leu Asp GIu Met GIn Leu Asp He GIn Ala Arg Tyr He Tyr 260 265 270

GIy Lys Pro VaI GIn GIy VaI Ala Tyr VaI Arg Phe GIy Leu Leu Asp 275 280 285

GIu Asp GIy Lys Lys Thr Phe Phe Arg GIy Leu GIu Ser GIn Thr Lys 290 295 300

Leu VaI Asn GIy GIn Ser His He Ser Leu Ser Lys Ala GIu Phe GIn 305 310 315 320

Asp Ala Leu GIu Lys Leu Asn Met GIy He Thr Asp Leu GIn GIy Leu 325 330 335

Arg Leu Tyr VaI Ala Ala Ala He He GIu Tyr Pro GIy GIy GIu Met 340 345 350 GIu GIu Ala GIu Leu Thr Ser Trp Tyr Pbe VaI Ser Ser Pro Phe Ser 355 360 365

Leu Asp Leu Ser Lys Thr Lys Arg His Leu VaI Pro GIy Ala Pro Phe 370 375 380

Leu Leu GIn Ala Leu VaI Arg GIu Met Ser GIy Ser Pro Ala Ser GIy 385 390 395 400

lie Pro VaI Lys VaI Ser Ala Thr VaI Ser Ser Pro GIy Ser VaI Pro

405 410 415

GIu VaI GIn Asp He GIn GIn Asn Thr Asp GIy Ser GIy GIn VaI Ser 420 425 430

He Pro He He He Pro GIn Thr He Ser GIu Leu GIn Leu Ser VaI 435 440 445

Ser Ala GIy Ser Pro His Pro Ala He Ala Arg Leu Thr VaI Ala Ala 450 455 460

Pro Pro Ser GIy GIy Pro GIy Phe Leu Ser He GIu Arg Pro Asp Ser 465 470 475 480

Arg Pro Pro Arg VaI GIy Asp Thr Leu Asn Leu Asn Leu Arg Ala VaI

485 490 495

GIy Ser GIy Ala Thr Phe Ser His Tyr Tyr Tyr Met He Leu Ser Arg 500 505 510

GIy GIn He VaI Phe Met Asn Arg GIu Pro Lys Arg Thr Leu Thr Ser 515 520 525

VaI Ser VaI Phe VaI Asp His His Leu Ala Pro Ser Phe Tyr Phe VaI 530 535 540

Ala Phe Tyr Tyr His GIy Asp His Pro VaI Ala Asn Ser Leu Arg VaI 545 550 555 560 Asp VaI GIn Ala GIy Ala Cys GIu GIy Lys Leu GIu Leu Ser VaI Asp 565 570 575

GIy Ala Lys GIn Tyr Arg Asn GIy GIu Ser VaI Lys Leu His Leu GIu 580 585 590

Thr Asp Ser Leu Ala Leu VaI Ala Leu GIy Ala Leu Asp Thr Ala Leu 595 600 605

Tyr Ala Ala GIy Ser Lys Ser His Lys Pro Leu Asn Met GIy Lys VaI 610 615 620

Phe GIu Ala Met Asn Ser Tyr Asp Leu GIy Cys GIy Pro GIy GIy GIy 625 630 635 640

Asp Ser Ala Leu GIn VaI Phe GIn Ala Ala GIy Leu Ala Phe Ser Asp 645 650 655

GIy Asp GIn Trp Thr Leu Ser Arg Lys Arg Leu Ser Cys Pro Lys GIu 660 665 670

Lys Thr Thr Arg Lys Lys Arg Asn VaI Asn Phe GIn Lys Ala lie Asn 675 680 685

GIu Lys Leu GIy GIn Tyr Ala Ser Pro Thr Ala Lys Arg Cys Cys GIn 690 695 700

Asp GIy VaI Thr Arg Leu Pro Met Met Arg Ser Cys GIu GIn Arg Ala 705 710 715 720

Ala Arg VaI GIn GIn Pro Asp Cys Arg GIu Pro Phe Leu Ser Cys Cys 725 730 735

GIn Phe Ala GIu Ser Leu Arg Lys Lys Ser Arg Asp Lys GIy GIn Ala 740 745 750

GIy Leu GIn Arg Ala Leu GIu lie Leu GIn GIu GIu Asp Leu lie Asp 755 750 765

GIu Asp Asp lie Pro VaI Arg Ser Phe Phe Pro GIu Asn Trp Leu Trp_, 770 775 780

Arg VaI GIu Thr VaI Asp Arg Phe GIn lie Leu Thr Leu Trp Leu Pro 785 790 795 800

Asp Ser Leu Thr Thr Trp GIu lie His GIy Leu Ser Leu Ser Lys Thr 805 810 815

Lys GIy Leu Cys VaI Ala Thr Pro VaI GIn Leu Arg VaI Phe Arg GIu 820 825 830

Phe His Leu His Leu Arg Leu Pro Met Ser VaI Arg Arg Phe GIu GIn 835 840 845

Leu GIu Leu Arg Pro VaI Leu Tyr Asn Tyr Leu Asp Lys Asn Leu Thr 850 855 860

VaI Ser VaI His VaI Ser Pro VaI GIu GIy Leu Cys Leu Ala GIy GIy 865 870 875 880

GIy GIy Leu Ala GIn GIn VaI Leu VaI Pro Ala GIy Ser Ala Arg Pro 885 890 895

VaI Ala Phe Ser VaI VaI Pro Thr Ala Ala Ala Ala VaI Ser Leu Lys 900 905 910

VaI VaI Ala Arg GIy Ser Phe GIu Phe Pro VaI GIy Asp Ala VaI Ser 915 920 925

Lys VaI Leu GIn lie GIu Lys GIu GIy Ala lie His Arg GIu GIu Leu 930 935 940

VaI Tyr GIu Leu Asn Pro Leu Asp His Arg GIy Arg Thr Leu GIu lie 945 950 955 960 Pro GIy Asn Ser A.sp Pro Asn Met Hs Pro Asp GIy Asp Phe Asn Ser 965 970 975

Tyr VaI Arg VaI Thr Ala Ser Asp Pro Leu Asp Thr Leu GIy Ser GIu 980 985 990

GIy Ala Leu Ser Pro GIy GIy VaI Ala Ser Leu Leu Arg Leu Pro Arg 995 1000 1005

GIy Cys GIy GIu GIn Thr Met lie Tyr Leu Ala Pro Thr Leu Ala 1010 1015 1020

Ala Ser Arg Tyr Leu Asp Lys Thr GIu GIn Trp Ser Thr Leu Pro 1025 1030 1035

Pro GIu Thr Lys Asp His Ala VaI Asp Leu lie GIn Lys GIy Tyr 1040 1045 1050

Met Arg He GIn GIn Phe Arg Lys Ala Asp GIy Ser Tyr Ala Ala 1055 1060 1065

Trp Leu Ser Arg GIy Ser Ser Thr Trp Leu Thr Ala Phe VaI Leu 1070 1075 1080

Lys VaI Leu Ser Leu Ala GIn GIu GIn VaI GIy GIy Ser Pro GIu 1085 1090 1095

Lys Leu GIn GIu Thr Ser Asn Trp Leu Leu Ser GIn GIn GIn Ala 1100 1105 1110

Asp GIy Ser Phe GIn Asp Pro Cys Pro VaI Leu Asp Arg Ser Met 1115 1120 1125

GIn GIy GIy Leu VaI GIy Asn Asp GIu Thr VaI Ala Leu Thr Ala 1130 1135 1140

Phe VaI Thr He Ala Leu His His GIy Leu Ala VaI Phe GIn Asp 1145 1150 1155 GIu GIy Ala GIu Pro Leu Lys GIn Arg VaI GIu Ala Ser lie Ser

1160 1165 1170

Lys Ala Asn Ser Phe Leu GIy GIu Lys Ala Ser Ala GIy Leu Leu

1175 1180 1185

GIy Ala His Ala Ala Ala lie Thr Ala Tyr Ala Leu Thr Leu Thr

1190 1195 1200

Lys Ala Pro VaI Asp Leu Leu GIy VaI Ala His Asn Asn Leu Met

1205 1210 1215

Ala Met Ala GIn GIu Thr GIy Asp Asn Leu Tyr Trp GIy Ser VaI 1220 1225 1230

Thr GIy Ser GIn Ser Asn Ala VaI Ser Pro Thr Pro Ala Pro Arg

1235 1240 1245

Asn Pro Ser Asp Pro Met Pro GIn Ala Pro Ala Leu Trp lie GIu

1250 1255 1260

Thr Thr Ala Tyr Ala Leu Leu His Leu Leu Leu His GIu GIy Lys

1265 1270 1275

Ala GIu Met Ala Asp GIn Ala Ala Ala Trp Leu Thr Arg GIn GIy

1280 1285 1290

Ser Phe GIn GIy GIy Phe Arg Ser Thr GIn Asp Thr VaI lie Ala 1295 1300 1305

Leu Asp Ala Leu Ser Ala Tyr Trp lie Ala Ser His Thr Thr GIu

1310 1315 1320

GIu Arg GIy Leu Asn VaI Thr Leu Ser Ser Thr GIy Arg Asn GIy

1325 1330 1335

Phe Lys Ser His Ala Leu GIn Leu Asn Asn Arg GIn lie Arg GIy 1340 1345 1350

Leu GIu GIu GIu Leu GIn Phe Ser Leu GIy Ser Lys lie Asn VaI

1355 1360 1365

Lys VaI GIy GIy Asn Ser Lys GIy Thr Leu Lys VaI Leu Arg Thr

1370 1375 1380

Tyr Asn VaI Leu Asp Met Lys Asn Thr Thr Cys GIn Asp Leu GIn 1385 1390 1395

lie GIu VaI Thr VaI Lys GIy His VaI GIu Tyr Thr Met GIu Ala

1400 1405 1410

Asn GIu Asp Tyr GIu Asp Tyr GIu Tyr Asp GIu Leu Pro Ala Lys

1415 1420 1425

Asp Asp Pro Asp Ala Pro Leu GIn Pro VaI Thr Pro Leu GIn Leu

1430 1435 ^' 1440

Phe GIu GIy Arg Arg Asn Arg Arg Arg Arg GIu Ala Pro Lys VaI

1445 1450 1455

VaI GIu GIu GIn GIu Ser Arg VaI His Tyr Thr VaI Cys lie Trp 1460 1465 1470

Arg Asn GIy Lys VaI GIy Leu Ser GIy Met Ala lie Ala Asp VaI

1475 1480 1485

Thr Leu Leu Ser GIy Phe His Ala Leu Arg Ala Asp Leu GIu Lys

1490 1495 1500

Leu Thr Ser Leu Ser Asp Arg Tyr VaI Ser His Phe GIu Thr GIu

1505 1510 1515

GIy Pro His VaI Leu Leu Tyr Phe Asp Ser VaI Pro Thr Ser Arg

1520 1525 1530 GIu Cys VaI GIy Phe GIu Ala Va. GIn GIu VaI Pro VaI GIy Leu 1535 1540 1545

VaI GIn Pro Ala Ser Ala Thr Leu Tyr Asp Tyr Tyr Asn Pro GIu 1550 1555 1560

Arg Arg Cys Ser VaI Phe Tyr GIy Ala Pro Ser Lys Ser Arg Leu 1565 1570 1575

Leu Ala Thr Leu Cys Ser Ala GIu VaI Cys GIn Cys Ala GIu GIy 1580 1585 1590

Lys Cys Pro Arg GIn Arg Arg Ala Leu GIu Arg GIy Leu GIn Asp 1595 1600 1605

GIu Asp GIy Tyr Arg Met Lys Phe Ala Cys Tyr Tyr Pro Arg VaI 1610 1615 1620

GIu Tyr GIy Phe GIn VaI Lys VaI Leu Arg GIu Asp Ser Arg Ala 1625 1630 1635

Ala Phe Arg Leu Phe GIu Thr Lys lie Thr GIn VaI Leu His Phe 1640 1645 1650

Thr Lys Asp VaI Lys Ala Ala Ala Asn GIn Met Arg Asn Phe Leu 1655 1660 1665

VaI Arg Ala Ser Cys Arg Leu Arg Leu GIu Pro GIy Lys GIu Tyr 1670 1675 1680

Leu lie Met GIy Leu Asp GIy Ala Thr Tyr Asp Leu GIu GIy His 1685 1690 1695

Pro GIn Tyr Leu Leu Asp Ser Asn Ser Trp lie GIu GIu Met Pro 1700 1705 1710

Ser GIu Arg Leu Cys Arg Ser Thr Arg GIn Arg Ala Ala Cys Ala 1715 1720 1725 GIn Leu Asn Asp Phe Leu GIn GIu Tyr GIy Thr GIn GIy Cys GIn 1730 1735 1740

VaI

<210> 20

<211> 1018 <212> PRT

<213> Artificial sequence

<220>

<223> contactin 1

<400> 20

Met Lys Met Trp Leu Leu VaI Ser His Leu VaI lie lie Ser lie Thr 1 5 10 15

Thr Cys Leu Ala GIu Phe Thr Trp Tyr Arg Arg Tyr GIy His GIy VaI 20 25 30

Ser GIu GIu Asp Lys GIy Phe GIy Pro lie Phe GIu GIu GIn Pro lie 35 40 45

Asn Thr lie Tyr Pro GIu GIu Ser Leu GIu GIy Lys VaI Ser Leu Asn 50 55 60

Cys Arg Ala Arg Ala Ser Pro Phe Pro VaI Tyr Lys Trp Arg Met Asn 65 70 75 80

Asn GIy Asp VaI Asp Leu Thr Ser Asp Arg Tyr Ser Met VaI GIy GIy 85 90 95

Asn Leu VaI lie Asn Asn Pro Asp Lys GIn Lys Asp Ala GIy lie Tyr 100 105 110 Tyr Cys Leυ Ala Ser Asn Asn Tyr GIy Met VaI Arg Ser Thr GIu Ala 115 120 125

Thr Leu Ser Phe GIy Tyr Leu Asp Pro Phe Pro Pro GIu GIu Arg Pro 130 135 140

GIu VaI Arg VaI Lys GIu GIy Lys GIy Met VaI Leu Leu Cys Asp Pro 145 150 155 160

Pro Tyr His Phe Pro Asp Asp Leu Ser Tyr Arg Trp Leu Leu Asn GIu

165 170 175

Phe Pro VaI Phe lie Thr Met Asp Lys Arg Arg Phe VaI Ser GIn Thr 180 185 190

Asn GIy Asn Leu Tyr lie Ala Asn VaI GIu Ala Ser Asp Lys GIy Asn 195 200 205

Tyr Ser Cys Phe VaI Ser Ser Pro Ser lie Thr Lys Ser VaI Phe Ser 210 215 220

Lys Phe He Pro Leu He Pro He Pro GIu Arg Thr Thr Lys Pro Tyr 225 230 235 240

Pro Ala Asp He VaI VaI GIn Phe Lys Asp VaI Tyr Ala Leu Met GIy

245 250 255

GIn Asn VaI Thr Leu GIu Cys Phe Ala Leu GIy Asn Pro VaI Pro Asp 260 265 270

He Arg Trp Arg Lys VaI Leu GIu Pro Met Pro Ser Thr Ala GIu He 275 280 285

Ser Thr Ser GIy Ala VaI Leu Lys He Phe Asn He GIn Leu GIu Asp 290 295 300

GIu GIy He Tyr GIu Cys GIu Ala GIu Asn He Arg GIy Lys Asp Lys 305 310 315 320 His GIn Ala Arg lie Tyr VaI GIn Ala Phe Pro GIu Trp VaI GIu His 325 330 335

lie Asn Asp Thr GIu VaI Asp lie GIy Ser Asp Leu Tyr Trp Pro Cys 340 345 350

VaI Ala Thr GIy Lys Pro lie Pro Thr lie Arg Trp Leu Lys Asn GIy 355 360 365

Tyr Ala Tyr His Lys GIy GIu Leu Arg Leu Tyr Asp VaI Thr Phe GIu 370 375 380

Asn Ala GIy Met Tyr GIn Cys lie Ala GIu Asn Thr Tyr GIy Ala lie 385 390 395 400

Tyr Ala Asn Ala GIu Leu Lys He Leu Ala Leu Ala Pro Thr Phe GIu 405 410 415

Met Asn Pro Met Lys Lys Lys He Leu Ala Ala Lys GIy GIy Arg VaI 420 425 430

He He GIu Cys Lys Pro Lys Ala Ala Pro Lys Pro Lys Phe Ser Trp 435 440 445

Ser Lys GIy Thr GIu Trp Leu VaI Asn Ser Ser Arg He Leu He Trp 450 455 460

GIu Asp GIy Ser Leu GIu He Asn Asn He Thr Arg Asn Asp GIy GIy 465 470 475 480

He Tyr Thr Cys Phe Ala GIu Asn Asn Arg GIy Lys Ala Asn Ser Thr 485 490 495

GIy Thr Leu VaI He Thr Asp Pro Thr Arg He He Leu Ala Pro He 500 505 510

Asn Ala Asp He Thr VaI GIy GIu Asn Ala Thr Met GIn Cys Ala Ala 515 520 525

Ser Phe Asp Pro Ala Leu Asp Leu Thr Phe VaI Trp Ser Phe Asn GIy 530 535 540

Tyr VaI lie Asp Phe Asn Lys GIu Asn lie His Tyr GIn Arg Asn Phe 545 550 555 560

Met Leu Asp Ser Asn GIy GIu Leu Leu lie Arg Asn Ala GIn Leu Lys 565 570 575

His Ala GIy Arg Tyr Thr Cys Thr Ala GIn Thr lie VaI Asp Asn Ser 580 585 590

Ser Ala Ser Ala Asp Leu VaI VaI Arg GIy Pro Pro GIy Pro Pro GIy 595 600 605

GIy Leu Arg lie GIu Asp lie Arg Ala Thr Ser VaI Ala Leu Thr Trp 610 615 620

Ser Arg GIy Ser Asp Asn His Ser Pro lie Ser Lys Tyr Thr lie GIn 625 630 635 640

Thr Lys Thr lie Leu Ser Asp Asp Trp Lys Asp Ala Lys Thr Asp Pro 645 650 655

Pro lie lie GIu GIy Asn Met GIu Ala Ala Arg Ala VaI Asp Leu lie 660 665 670

Pro Trp Met GIu Tyr GIu Phe Arg VaI VaI Ala Thr Asn Thr Leu GIy 675 680 685

Arg GIy GIu Pro Ser lie Pro Ser Asn Arg lie Lys Thr Asp GIy Ala 690 695 700

Ala Pro Asn VaI Ala Pro Ser Asp VaI GIy GIy GIy GIy GIy Arg Asn 705 710 715 720 Arg GIu Leu Thr He Thr Trp Ala Pro Leu Ser Arg GIu Tyr His Tyr 725 730 735

GIy Asn Asn Phe GIy Tyr He VaI Ala Phe Lys Pro Phe Asp GIy GIu 740 745 750

GIu Trp Lys Lys VaI Thr VaI Thr Asn Pro Asp Thr GIy Arg Tyr VaI 755 760 765

His Lys Asp GIu Thr Met Ser Pro Ser Thr Ala Phe GIn VaI Lys VaI 770 775 780

Lys Ala Phe Asn Asn Lys GIy Asp GIy Pro Tyr Ser Leu Leu Ala VaI 785 790 795 800

He Asn Ser Ala GIn Asp Ala Pro Ser GIu Ala Pro Thr GIu VaI GIy 805 810 815

VaI Lys VaI Leu Ser Ser Ser GIu He Ser VaI His Trp GIu His VaI 820 825 830

Leu GIu Lys He VaI GIu Ser Tyr GIn He Arg Tyr Trp Ala Ala His 835 840 845

Asp Lys GIu GIu Ala Ala Asn Arg VaI GIn VaI Thr Ser GIn GIu Tyr 850 855 860

Ser Ala Arg Leu GIu Asn Leu Leu Pro Asp Thr GIn Tyr Phe He GIu 865 870 875 880

VaI GIy Ala Cys Asn Ser Ala GIy Cys GIy Pro Pro Ser Asp Met He 885 890 895

GIu Ala Phe Thr Lys Lys Ala Pro Pro Ser GIn Pro Pro Arg He He 900 905 910

Ser Ser VaI Arg Ser GIy Ser Arg Tyr He He Thr Trp Asp His VaI 915 920 925 VaI Ala Leu Ser Asn GIu Ser Thr VaI Thr GIy Tyr Lys VaI Leu Tyr 930 935 940

Arg Pro Asp GIy GIn His Asp GIy Lys Leu Tyr Ser Thr His Lys His 945 950 955 960

Ser lie GIu VaI Pro lie Pro Arg Asp GIy GIu Tyr VaI VaI GIu VaI 965 970 975

Arg Ala His Ser Asp GIy GIy Asp GIy VaI VaI Ser GIn VaI Lys lie 980 985 990

Ser GIy Ala Pro Thr Leu Ser Pro Ser Leu Leu GIy Leu Leu Leu Pro 995 1000 1005

Ala Phe GIy lie Leu VaI Tyr Leu GIu Phe 1010 1015

<210> 21

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<223> neuronal pentraxin receptor

<400> 21

Met Leu Ala Phe Leu GIy Ala VaI He Cys lie He Ala Ser VaI Pro 1 5 10 15

Leu Ala Ala Ser Pro Ala Arg Ala Leu Pro GIy GIy Ala Asp Asn Ala 20 25 30

Ser VaI Ala Ser GIy Ala Ala Ala Ser Pro GIy Pro GIn Arg Ser Leu 35 40 45 Ser Ala Leu His GIy Ala GIy GIy Ser Ala GIy Pro Pro Ala Leu Pro 50 55 60

GIy Ala Pro Ala Ala Ser Ala His Pro Leu Pro Pro GIy Pro Leu Phe 65 70 75 80

Ser Arg Phe Leu Cys Thr Pro Leu Ala Ala Ala Cys Pro Ser GIy Ala 85 90 95

GIn GIn GIy Asp Ala Ala GIy Ala Ala Pro GIy GIu Arg GIu GIu Leu 100 105 110

Leu Leu Leu GIn Ser Thr Ala GIu GIn Leu Arg GIn Thr Ala Leu GIn 115 120 125

Gin GIu Ala Arg lie Arg Ala Asp GIn Asp Thr lie Arg GIu Leu Thr 130 135 140

GIy Lys Leu GIy Arg Cys GIu Ser GIy Leu Pro Arg GIy Leu GIn GIy 145 150 155 160

Ala GIy Pro Arg Arg Asp Thr Met Ala Asp GIy Pro Trp Asp Ser Pro 165 170 175

Ala Leu lie Leu GIu Leu GIu Asp Ala VaI Arg Ala Leu Arg Asp Arg 180 185 190

lie Asp Arg Leu GIu GIu Leu Pro Ala Arg VaI Asn Leu Ser Ala Ala 195 200 205

Pro Ala Pro VaI Ser Ala VaI Pro Thr GIy Leu His Ser Lys Met Asp 210 215 220

GIn Leu GIu GIy GIn Leu Leu Ala GIn VaI Leu Ala Leu GIu Lys GIu 225 230 235 240

Arg VaI Ala Leu Ser His Ser Ser Arg Arg GIn Arg GIn GIu VaI GIu 245 250 255 Lys GIu Leu Asp VaI Leu GIn GIy Arg VaI Ala GIu Leu GIu His GIy 260 265 270

Ser Ser Ala Tyr Ser Pro Pro Asp Ala Phe Lys lie Ser lie Pro lie 275 280 285

Arg Asn Asn Tyr Met Tyr Ala Arg VaI Arg Lys Ala Leu Pro GIu Leu 290 295 300

Tyr Ala Phe Thr Ala Cys Met Trp Leu Arg Ser Arg Ser Ser GIy Thr 305 310 315 320

GIy GIn GIy Thr Pro Phe Ser Tyr Ser VaI Pro GIy GIn Ala Asn GIu 325 330 335

lie VaI Leu Leu GIu Ala GIy His GIu Pro Met GIu Leu Leu lie Asn 340 345 350

Asp Lys VaI Ala GIn Leu Pro Leu Ser Leu Lys Asp Asn GIy Trp His 355 360 365

His lie Cys lie Ala Trp Thr Thr Arg Asp GIy Leu Trp Ser Ala Tyr 370 375 380

GIn Asp GIy GIu Leu GIn GIy Ser GIy GIu Asn Leu Ala Ala Trp His 385 390 395 400

Pro lie Lys Pro His GIy lie Leu lie Leu GIy GIn GIu GIn Asp Thr 405 410 415

Leu GIy GIy Arg Phe Asp Ala Thr GIn Ala Phe VaI GIy Asp lie Ala 420 425 430

GIn Phe Asn Leu Trp Asp His Ala Leu Thr Pro Ala GIn VaI Leu GIy 435 440 445

lie Ala Asn Cys Thr Ala Pro Leu Leu GIy Asn VaI Leu Pro Trp GIu 450 455 460

Asp Lys Leu VaI GIu Ala Phe GIy GIy Ala Thr Lys Ala Ala Phe Asp 465 470 475 480

VaI Cys Lys GIy Arg Ala Lys Ala 485

<210> 22 <211> 266

<212> PRT

<213> Artificial sequence

<220> <223> RNA binding motif protein 7

<400> 22

Met GIy Ala Ala Ala Ala GIu Ala Asp Arg Thr Leu Phe VaI GIy Asn 1 5 10 15

Leu GIu Thr Lys VaI Thr GIu GIu Leu Leu Phe GIu Leu Phe His GIn 20 25 30

Ala GIy Pro VaI lie Lys VaI Lys He Pro Lys Asp Lys Asp GIy Lys 35 40 45

Pro Lys GIn Phe Ala Phe VaI Asn Phe Lys His GIu VaI Ser VaI Pro 50 55 60

Tyr Ala Met Asn Leu Leu Asn GIy He Lys Leu Tyr GIy Arg Pro He 65 70 75 80

Lys He GIn Phe Arg Ser GIy Ser Ser His Ala Pro GIn Asp VaI Ser 85 90 95

Leu Ser Tyr Pro GIn His His VaI GIy Asn Ser Ser Pro Thr Ser Thr 100 ' 105 HO Ser Pro Ser Arg Tyr GIu Arg Thr Met Asp Asn Met Thr Ser Ser Ala 115 120 125

GIn lie lie GIn Arg Ser Phe Ser Ser Pro GIu Asn Phe GIn Arg GIn 130 135 140

Ala VaI Met Asn Ser Ala Leu Arg GIn Met Ser Tyr GIy GIy Lys Phe 145 150 155 160

GIy Ser Ser Pro Leu Asp GIn Ser GIy Phe Ser Pro Ser VaI GIn Ser 165 170 175

His Ser His Ser Phe Asn GIn Ser Ser Ser Ser GIn Trp Arg GIn GIy 180 185 190

Thr Pro Ser Ser GIn Arg Lys VaI Arg Met Asn Ser Tyr Pro Tyr Leu 195 200 205

Ala Asp Arg His Tyr Ser Arg GIu GIn Arg Tyr Thr Asp His GIy Ser 210 215 220

Asp His His Tyr Arg GIy Lys Arg Asp Asp Phe Phe Tyr GIu Asp Arg 225 230 235 240

Asn His Asp Asp Trp Ser His Asp Tyr Asp Asn Arg Arg Asp Ser Ser 245 250 255

Arg Asp GIy Lys Trp Arg Ser Ser Arg His 260 265

<210> 23

<211> 109

<212> PRT <213> Artificial sequence

<220>

<223> Ig kappa chain V-III region SIE <400> 23

GIu lie VaI Leu Thr GIn Ser Pro GIy Thr Leu Ser Leu Ser Pro GIy 1 5 10 15

GIu Arg Ala Thr Leu Ser Cys Arg Ala Ser GIn Ser VaI Ser Asn Ser 20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Lys Pro GIy GIn Ala Pro Arg Leu Leu 35 40 45

lie Tyr GIy Ala Ser Ser Arg Ala Thr GIy lie Pro Asp Arg Phe Ser 50 55 60

GIy Ser GIy Ser GIy Thr Asp Phe Thr Leu Thr lie Ser Arg Leu GIu 65 70 75 80

Pro Asp Asp Phe Ala VaI Tyr Tyr Cys GIn GIn Tyr GIy Ser Ser Pro 85 90 95

GIn Thr Phe GIy GIn GIy Ser Lys VaI GIu lie Lys Arg 100 105

Claims

Claims

1. A method for detecting neurodegenerative disease in a subject, comprising determining the expression level of the protein chromogranin A in blood, cerebrospinal fluid or nervous tissue.

2. Method according to claim 1, wherein said expression level is determined in a sample of blood, cerebrospinal fluid or nervous tissue of said subject.

3. Method according to claim 1 or 2, wherein said neurodegenerative disease is multiple sclerosis or CIS.

4. Method according to any one of claims 1-3, wherein in addition to said expression level of chromogranin A, also the expression level of one or more of the proteins selected from, clusterin, complement C3, complement C4B, beta V spectrin, osteopontin, apolipoprotein D, complement C4A, contactin 1, neuronal pentraxin receptor, RNA binding motif protein and Ig kappa chain V-III region SIE is determined.

5. Method according to any one of claims 1-4, wherein said expression level is determined by detecting the said protein or a peptide fragment thereof in a mass range of 800 to 27,000 Da.

6. Method according to any one of the preceding claims, wherein said detection is performed by immunological assay or mass spectrometry.

7. Use of a method according to any one of claims 1-6, wherein said use is for monitoring a disease process or a response to a disease therapy.

8. Use according to claim 7, wherein said disease is multiple sclerosis or CIS.

9. Marker protein or marker peptide for detecting neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject wherein said marker protein is the expression level of the protein chromogranin A in blood, cerebrospinal fluid or nervous tissue and wherein said marker peptide is a peptide fragment of chromogranin A having a mass of between 800 and 27,000 Da.

10. Marker profile for detecting neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject wherein said marker profile comprises the expression level in blood, cerebrospinal fluid or nervous tissue of a subject of a first protein being chromogranin A or a peptide thereof, and wherein said marker profile further comprises at least one additional expression level of a protein or peptide fragment selected from the group of clusterin, complement 03, complement C4B, beta V spectrin, osteopontin, apolipoprotein D, complement C4A, contactin 1, neuronal pentraxin receptor, RNA binding motif protein and Ig kappa chain V-III region SIE.

11. Use of a marker protein or marker profile as defined in claim 9 or 10, for detecting neurodegenerative disease, preferably multiple sclerosis or CIS, in a subject.