Human reticulocalbin isoforms

Details Human reticulocalbin isoforms Human reticulocalbin isoforms

1999-03-16

2001-05-22

Low, Chirstopher S. F. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S069100, C435S320100, C435S325000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C536S063000, C536S022100, C536S023500

Reexamination Certificate

active

06235477

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of human reticulocalbins and to the use of these sequences in the diagnosis, prevention, and treatment of infectious, developmental, neoplastic, and immunological disorders.
BACKGROUND OF THE INVENTION
The endoplasmic reticulum (ER) is a system of intracellular membranes in which protein synthesis and other important metabolic processes take place. In cells where the major function of the endoplasmic reticulum is protein synthesis, this organelle contains a large number of ribosomes and is known as the rough endoplasmic reticulum. Endoplasmic reticulum that is engaged primarily in steroid hormone biosynthesis and contains few ribosomes is known as the smooth endoplasmic reticulum.
The ER also serves as an intracellular store of Ca
2+
([Ca
2
+]
i
). Following stimulation by second messenger molecules, such as inositoltrisphosphate, [Ca
2
+]
i
is briefly released from the ER into the surrounding cytoplasm. Increased levels of [Ca
2
+]
i
in the cytoplasm activate a number of enzymatic processes, some of which contribute to cell cycle events, and/or to cellular differentiation. Similar processes take place in the dividing cell nucleus during breakdown of the nuclear membrane and segregation of chromatids at anaphase.
In muscle, the ER is termed the sarcoplasmic reticulum (SR) and is the principal source of [Ca
2
+]
i
, which drives muscle contraction. [Ca
2
+]
i
binds to calmodulin (CaM), which activates CaM protein kinase. CaM protein kinase then phosphorylates light-chain myosin. In relaxed muscle, myosin is prevented from interacting with actin by tropomyosin. Ca
2+
binds tropomyosin, causing a conformational change that leads to the release of actin. Phosphorylated myosin interacts with actin, forming actinomyosin, and the contraction process is initiated. Muscle relaxation is brought about by active transport of Ca
2+
into the SR by a calcium ATPase pump.
The calcium-binding domain of many proteins contains the high affinity Ca
2+
-binding motif often referred to as the EF-hand (Kretsinger, R. H. and Nockolds, C. E. (1973) J. Biol. Chem. 248:3313-3326). The EF-hand is characterized by a twelve amino acid residue-containing loop, flanked by two &agr;-helices, orientated approximately 90° with respect to one another. Aspartate (D) and glutamate (E) residues are usually found at positions 10 and 21, respectively, bordering the twelve amino acid loop. In addition, a conserved glycine residue in the central portion of the loop is found in most Ca
2+
-binding EF-hand domains. Oxygen ligands within this domain coordinate the Ca
2+
ion.
Numerous soluble proteins are retained within the ER by a specific retrieval receptor which recognizes a C-terminal tetrapeptide, defined as Lys/His-Asp-Glu-Leu (K/HDEL). ER soluble proteins include endoplasmin, BiP, PDI, and calrecticulin. Calrecticulin is believed to be the major Ca
2+
-storage protein of the ER; the other three proteins are involved in folding and maturation of secretory proteins. All four proteins bind Ca
2+
, but none are members of the EF-hand family (Weis, K. et al. (1994) J. Biol. Chem. 269:19142-19150).
Novel endoplasmic reticulum Ca
2+
-binding proteins have also been identified. Human reticulocalbin is an ER luminal protein isolated from a transitional carcinoma cell line. The protein has six repeats of a domain containing the EF-hand domain, an HDEL C-terminal tetrapeptide, and binds Ca
2+
. A conserved glycine residue in the central portion of three of the EF-hand domains is absent, suggesting that human reticulocalbin plays some role(s) besides Ca
2+
-binding (Ozawa, M. (1995) J. Biochem. (Tokyo) 117:1113-1119). A similar protein, ERC-55, has been isolated from HeLa cells. It has six EF-hand repeats and HDEL C-terminal peptide, and binds Ca
2+
. The conserved glycine residue is absent from three of the ERC-55 EF-hand domains (Weis, K. et al. (supra)). Both proteins are expressed ubiquitously, particularly in heart, placenta, lung, and skeletal muscle (human reticulocalbin) and in kidney and skeletal muscle (ERC-55) (Ozawa, M. and Muramatsu, T. (supra); Weis, K. et al. (supra)).
The human gene encoding human reticulocalbin has been localized to a region on chromosome 11 (11p13). The gene is hemizygously deleted in individuals with the Wilms tumor, aniridia, genitourinary anomalies, mental retardation (WAGR) syndrome. The homologous murine reticulocalbin gene maps to a region of conserved synteny on mouse chromosome 2 and is deleted in the Small eye Harwell (Sey
H
) mutation. Loss of the murine reticulocalbin gene could contribute to the early lethality of Sey
H
and Sey
Dey
homozygotes (Kent, J. et al. (1997) Genomics 42:260-267).
Overexpression of reticulocalbin mRNA has been associated with the increased matrigel invasive properties of three human breast cancer cell lines. Conversely, reticulocalbin was not found to be expressed in two poorly invasive breast cancer cell lines (Liu, Z. et al. (1997) Biochem. Biophys. Res. Comm. 231:283-289).
An endogenous monoclonal antibody (MAb), which promotes central nervous system remyelination in a mouse model of multiple sclerosis, specifically reacts with nine independent neonatal rat brain cDNA clones. Five of the clones are identical or highly similar to known cDNAs or proteins. One of the unknown clones (REM#1) encodes a 98 amino acid truncated protein (Asakura, K. et al. (1996) J. Neuroimmunol. 65:11-19). The MAb immunostains brain, spinal cord, heart, liver, kidney, stomach, erythrocytes, and small intestine. In particular, immunoreactivity is observed for both surface and cytoplasmic determinants in glial cells and in dendritic cells of the spleen, thymus, and lymph node (Miller, D. J. et al. (1994) J. Neurosci. 14:6230-6238).
The discovery of new human reticulocalbin isoforms and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of infectious, developmental, neoplastic, and immunological disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human reticulocalbin isoforms (designated collectively as RCN and individually as RCN &ggr; and RCN &dgr;) having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, respectively, or fragments thereof
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at le

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