Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1990-01-19
1993-11-23
Cashion, Jr., Merrell C.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530395, 530350, 530324, 435 691, 424 858, C07K 1300, C07K 900, C07K 1514, A61K 3900
Patent
active
052645541
DESCRIPTION:
BRIEF SUMMARY
ecule that encodes a PECAM variant. In one preferred embodiment, a polynucleotide molecule encodes a protein, preferably having a polypeptide molecular weight of about 79 kilodaltons (kd), with a N-terminus Gln-Glu-Asn-Ser-Phe-Thr-Ile and, moreover, hybridizes under high stringency conditions with an oligonucleotide probe that is complementary to a portion, at least about 10 nucleotides in length, of the nucleotide sequence depicted in FIG. 1.
An oligonucleotide is that additionally provided that corresponds to or is complementary to a portion of a nucleotide sequence depicted in FIG. 1 such that the presence of the oligonucleotide, or a transcription product thereof, in a cell expressing a DNA sequence which encodes PECAM-1 or a PECAM-1 variant, inhibits transcription or translation of the DNA sequence. In preferred embodiments, the oligonucleotide either is RNA (and inhibits translation of the DNA sequence) or is DNA (and inhibits transcription of said DNA sequence).
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Unless otherwise indicated, the respective contents of the documents cited below are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1D shows the primary structure of a platelet membrane glycoprotein within the present invention (PECAM-1), along with the nucleotide sequence encoding that primary structure. The hydrophobic putative signal peptide and transmembrane domain of the mature glycoprotein are underlined. Two naturally occurring Eco Rl sites are boxed. Cysteine residues, spaced approximately 50 amino acids apart throughout the entire external domain of PECAM-1, are circled; these are thought to participate in disulfide-bond formation within individual immunoglobulin homology units. Predicted glycosylation sites are designated with black triangles. A potential tyrosine phosphorylation site at residue 686 is indicated with a closed circle. The TAG stop codon is underlined in bold.
FIG. 2 is a line drawing schematically depicting PECAM-1, along with several other CAM molecules mentioned above, in relation to the cell membrane (stippled region). The circles are representations of individual immunoglobulin homology units. According to the classification of Williams, Immunology Today 8: 298 (1987), the depicted CAMs are made up of C2-type domains, with the exception of CEA, which has one V-type domain at the amino terminus. In fasciclin II and N-CAM, the hatched boxes proximal to the membrane represent fibronectin type III domains. Putative N-linked carbohydrate chains on PECAM-1 are symbolized by a ( ).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A platelet membrane glycoprotein has been discovered that is properly characterized as a CAM, according to the criteria discussed above. The mature protein, lacking a 27 amino-acid signal peptide sequence, has a molecular weight of about 130 kilodaltons (kd). Although this size is in the same range as a number of known platelet membrane glycoproteins, the new glycoprotein can be obtained in recoverable amounts, pursuant to the present invention, in a form such that the glycoprotein preparation migrates as a single band on a silver stained SDS-PAGE gel ("substantially pure form"). Preferably, the glycoprotein is in a form of sufficient purity to be usable in therapeutic contexts, including those discussed below.
The glycoprotein of the present invention is designated Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) because it is a CAM found on both platelet and endothelial cell membranes. In mature form, PECAM-1 has a 711 amino-acid sequence (polypeptide molecular weight: 79,578 dal
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The United States Government has certain rights in the invention described and claimed herein as a result of support, under grants from the United States National Institutes of Health, of work related to said invention.
Cashion Jr. Merrell C.
Perkins Susan M.
The Blood Center of Southeastern Wisconsin, Inc.
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