Human NESP55 polypeptides, polynucleotides and uses thereof

Details Human NESP55 polypeptides, polynucleotides and uses thereof Human NESP55 polypeptides, polynucleotides and uses thereof

1999-12-21

2003-08-19

Kunz, Gary (Department: 1647)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S016700, C514S018700, C530S300000, C530S328000, C530S330000, C530S350000

Reexamination Certificate

active

06608025

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to polypeptides, polynucleotides and uses thereof in screening and in medicine.
BACKGROUND OF THE INVENTION
Obesity is a serious health hazard that may be accompanied by elevated levels of blood cholesterol and elevated blood pressure. There is an increased risk of mortality from coronary heart disease, stroke, certain cancers and non-insulin dependent diabetes, as well as an association with several non-fatal health problems.
Obesity is usually measured by Body Mass Index (BMI), which is obtained from a simple formula, given by:
BMI
=(mass/kg)×(height/m)
−2
BMI values in adults have been categorised according to the severity of health risk, with the higher graded numbers associated with a more severe risk (Table 1). Modified versions of this scale have been derived which account for gender, and there are other considerations such as age and body frame (see, for example Black's Medical Dictionary).
TABLE 1
Grade
BMI
Category
3
>40
Severely obese
2
30-40
Obese
1
25-29.9
Overweight
0
20-24.9
Desirable weight
Ungraded
<20
Underweight
Desirable body weights for adults according to gender, height and build are given in Appendix 6C of Black's Medical Dictionary.
Ischia et al (1997)
J Biol Chem
272, 11657-11662 describes the cloning of a bovine chromogranin-like polypeptide termed NESP55 (neuroendocrine secretory protein of Mol Wt 55,000). A partial sequence for mouse NESP55 is also presented. As reviewed by Ischia et al, chromogranins are proteins found in the content of large dense core vesicles, specialised vesicular containers found in presynaptic terminals which store neurotransmitters and peptides prior to exocytic release during synaptic transmission. Chromogranins have an acid pI of 4 to 5 and typically consist of 200-700 amino acids with glutamic acid as the most abundant individual amino acid. Multiple pairs of consecutive basic amino acid residues, known as potential cleavage sites for trypsin-like endoproteases, are present. Both intracellular (involvement in sorting of peptidergic components to the large dense core vesicles) as well as extracellular functions (representing precursors of small biologically active neuropeptides such as pancreastatin, vasostatin or secretoneurin) have been proposed for chromogranins.
Bovine NESP55 is proposed to be a precursor for the tetrapeptide Leu-Ser-Ala-Leu (SEQ ID NO: 3), which has been identified as an endogenous antagonist of the serotonergic 5-HT
1B
receptor subtype. The serotonergic system is thought to play a role in mental disorders, particularly depression. A second amino acid sequence, GAIPIRRH (SEQ ID NO: 4), present at the C-terminus of bovine NESP55 is the same as that of a peptide identified in the secretory content of chromaffin granules (Sigafoos et al (1993)
J Anat
183, 253-264). A function was not assigned to this peptide, and there has been no suggestion that either peptide is involved in obesity.
SUMMARY OF THE INVENTION
We have identified a human homologue of the bovine NESP55 gene. Further, we have found that NESP55 is, at least in humans, linked with obesity. Accordingly, the invention provides isolated human neuroendocrine secretory protein 55 (NESP55).
In one aspect, the invention provides a substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative, wherein the polypeptide variant has an amino acid sequence which has at least 90% identity with the amino acid sequence of SEQ ID NO: 2. The invention also provides a processed polypeptide derivable from human NESP55 wherein the processed polypeptide is derivable, or predicted from the amino acid sequence of human NESP55 to be derivable, by endoproteolytic cleavage of human NESP55. In one embodiment, the N-terminal amino acid residue of the processed polypeptide is immediately preceded in the amino acid sequence of human NESP55 by two consecutive basic amino acid residues or by a basic amino acid residue. In another embodiment, the C-terminus of the processed polypeptide is immediately preceded by two consecutive basic amino acid residues or by a basic amino acid residue. In preferred embodiments, the processed polypeptide comprises the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6), or consists of the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6).
The invention also provides a polypeptide consisting of the amino acid sequence X
n
LHALZ
m
(SEQ ID NO: 11), or X
n
GPIPIRRHZ
m
(SEQ ID NO: 12) wherein X
n
represents the amino acid sequence of the consecutive n amino acids immediately N terminal to the amino acid sequence LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6) and wherein Z
m
represents the amino acid sequence of the consecutive m amino acids immediately C terminal to the amino acid sequence LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6), wherein n and m may independently be any number between 0 and 30 amino acids. In one embodiment, one or both of X
n
or Z
m
consists of the sequence immediately flanking the LHAL (SEQ ID NO: 5) or GPIPIRRH (SEQ ID NO: 6) sequences in native human NESP55.
15. The invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, as well as isolated polypeptides comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 2, or comprising an amino acid sequence at least 99.5% identical to the amino acid sequence of SEQ ID NO: 2. Isolated peptides consisting of the amino acid sequence LHAL (SEQ ID NO: 5) or the amino acid sequence GPIPIRRH (SEQ ID NO: 6) are also provided.
Another aspect of the invention pertains to isolated polynucleotides encoding or complementary to a polynucleotide encoding the polypeptides of the invention, e.g. encoding human NESP55 or encoding SEQ ID NO: 2. In a preferred embodiment, the isolated polynucleotide comprises the nucleotide of SEQ ID NO: 1. In another preferred embodiment, the polynucleotide is suitable for expressing the polypeptide of the invention in a host cell. The invention further provides vector constructs comprising the polynucleotides of the invention, and host cells transformed with the vector constructs of the invention. Still further, methods of making human NESP55 polypeptide, comprising culturing a host cell of the invention and isolating the human NESP55 polypeptide from the host cell, or culture medium, are also provided. Polypeptides obtainable by these methods are also encompassed by the invention.
Yet another aspect of the invention pertains to antibodies reactive towards the polypeptides of the invention. In one embodiment, the antibody is reactive towards the peptide sequence LHAL (SEQ ID NO: 5) and GAIPIPIRRH (SEQ ID NO: 6).
Pharmaceutical compositions, comprising an antibody of the invention, a NESP55 polypeptide of the invention or a NESP55 processed polypeptide of the invention, as well as a pharmaceutically acceptable carrier, are also provided. Methods of treating or preventing obesity in a patient, the comprising administering to the patient an effective amount of an antibody of the invention, a NESP55 polypeptide of the invention, a NESP55 processed polypeptide of the invention or a pharmaceutical composition of the invention are also provided.
Another aspect of the invention pertains to a method of identifying a polypeptide (interacting polypeptide) that is capable of interacting with a human NESP55, or a processed polypeptide thereof. The method comprises the steps of (1) exposing the polypeptide to a test composition that may comprise an interacting polypeptide, (2) detecting an interaction between the polypeptide and an interacting polypeptide and optionally (3) identifying a

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