Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-04-25
2004-05-25
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S320100, C435S471000, C435S252300, C435S325000, C536S023500, C530S350000
Reexamination Certificate
active
06740504
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of secreted proteins that are related to the transcobalamin II secreted subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
Secreted Proteins
Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. The present invention advances the state of the art by providing many novel human secreted proteins.
Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a ‘signal sequence trap’ technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer.
Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth. Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al.,
Mutat Res
1991 March-November; 256(2-6): 127-38).
The secreted form of amyloid beta/A4 protein precursor (APP) functions as a growth and/or differentiation factor. The secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms. (Roch et al.,
Ann N Y Acad Sci
Sep. 24, 1993;695:149-57). Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. The prominent effects of secreted APPs on synaptogenesis and neuronal survival suggest that secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson et al.,
Perspect Dev Neurobiol
1998; 5(4):337-52).
Breast cancer cells secrete a 52K estrogen-regulated protein (see Rochefort et al.,
Ann N Y Acad Sci
1986;464:190-201). This secreted protein is therefore useful in breast cancer diagnosis.
Two secreted proteins released by platelets, platelet factor 4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators of platelet involvement in hemostasis and thrombosis and assays that measure these secreted proteins are useful for studying the pathogenesis and course of thromboembolic disorders (Kaplan,
Adv Exp Med Biol
1978;102:105-19).
Vascular endothelial growth factor (VEGF) is another example of a naturally secreted protein. VEGF binds to cell-surface heparan sulfates, is generated by hypoxic endothelial cells, reduces apoptosis, and binds to high-affinity receptors that are up-regulated by hypoxia (Asahara et al.,
Semin Interv Cardiol
1996 September;1(3):225-32).
Many critical components of the immune system are secreted proteins, such as antibodies, and many important functions of the immune system are dependent upon the action of secreted proteins. For example, Saxon et al.,
Biochem Soc Trans
1997 May;25(2):383-7, discusses secreted IgE proteins.
For a further review of secreted proteins, see Nilsen-Hamilton et al.,
Cell Biol Int Rep
1982 September;6(9):815-36.
Transcobalamin II
Many biochemical reactions require the involvement of cobalamin (“Cbl”), also known as vitamin B12, as coenzyme factors. Human Cbl-dependent metabolism includes the biosynthesis of methionine from homocysteine and the isomerization of methylmalonyl-CoA to succinyl-CoA. Although cobalamin is highly water-soluble, it is nevertheless impervious to plasma membrane. Cobalamin is delivered into the designated subcellular locations through multiple physiological steps.
The cellular uptake of cobalamin is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TC II and, therefore, served as the source of the library from which the TC II cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5′-untranslated segment of 37 nucleotides, and a 3′-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene. (Platica, et al., J Biol Chem Apr 25;266(12):7860-3 (1991))
Extracellularly secreted cobalamin is continually transported across cellular space through transcytosis within the endomembrane-secretory system, within which transcobalamin II (“TC II”) binds to proteolytically-released Cbl. TC II-Cbl-containing vesicles release their contents into the circulation system. The uptake of TC II-Cbl from the circulating fluids utilizes similar pathways, including receptor-mediated translocation, vesicle-dependent trafficking and targeting, and lysosome-based proteolytical release.
TC II is a non-glycosylated secretory protein of molecular mass 43 kDa in plasma while its homologs IF and haptocorrin are heavily glycosylated. A conserved Cbl-binding domain (ProSite pattern: PS000468) exists among the three types of the proteins (Seetharam and Li, Vitam. Horm. 59:337-366 (2000); Seetharam B, et al., Annu. Rev. Nutr. 19:173-195 (1999); Hofman, et al., Nuc. Acid Res. 27: 215-219 (1999)). The affinity toward Cbl is suggested to be the highest for haptocorrin (Fedosov, et al., Biochim. Biophys. Acta 1292:113-119 (1996)). Of two TCs, TC I has been identified as a major protein constituent of secondary granules in neotrophil and mapped onto chromosome 11q11-q12 (Johnston, et al., J. Biol. Ch
Beasley Ellen M
Gong Fangcheng
Higgins Maureen E
Ladunga Steven I
Wei Ming-Hui
Applera Corporation
Celera Genomics
Hamud Fozia
Karjala Justin D.
Kunz Gary
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