Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-08-02
1998-07-21
Kemmerer, Elizabeth C.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 711, 43525411, 43525421, 4352543, 514 8, 530395, C12N 1563, C12N 115, A61K 3817, C07K 1447
Patent
active
057834164
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is filed under 35 USC 371 as the national stage of International Application PCT/DK94/00037 filed Jan. 20, 1994, which is incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to human spasmolytic polypeptide in glycosylated form, variants of human and porcine spasmolytic polypeptides and a method of producing spasmolytic polypeptides in glycosylated form.
BACKGROUND OF THE INVENTION
Human spasmolytic polypeptide (HSP) belongs to a family of peptides domain is made up of a sequence of 38 or 39 amino acid residues in which 6 cystein residues are linked in the configuration 1-5, 2-4 and 3-6 thus (FIG. 1).
The physiological function of the trefoil peptides is poorly understood, and so far only PSP has been studied in any detail. In the porcine pancreas, PSP is found in the acinar cells and to be secreted in large amounts (50-100 mg/ml) into the pancreatic juice upon stimulation with binding of PSP to rat intestinal mucosa cells and membrane preparations gastrointestinal tract, specific receptor-like binding to Paneth cells in intraluminal function of the peptide. A pharmacological screening has indicated that PSP has spasmolytic and gastric acid secretion inhibitory
The DNA sequence and derived amino acid sequence of the human counterpart found to be expressed in the stomach, but not in the pancreas to any reported to be associated with peptic ulcers and mucosal injury in of these peptides.
Only very limited amounts of HSP can be prepared by extraction of human tissue. An object of study resulting in the present invention was therefore to prepare recombinant HSP in sufficient amounts for physiological and biochemical studies of the peptide.
SUMMARY OF THE INVENTION
It has surprisingly been found that when recombinant HSP is produced in certain host organisms, a proportion of it is produced in glycosylated form by posttranslational modifications. The glycosylated form of HSP has not, to applicant's best knowledge, been described previously.
Accordingly, the present invention relates to human spasmolytic polypeptide (HSP) which has the amino acid sequence Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Asp Asn Gly Cys Cys Phe Asp Ser Ser Val Thr Gly Val Pro Trp Cys Phe His Pro Leu Pro Lys Gln Glu Ser Asp Gln Cys Val Met Glu Val Ser Asp Arg Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu Cys Ala Ser Arg Lys Cys Cys Phe Ser Asn Phe Ile Phe Glu Val Pro Trp Cys Phe Phe Pro Asn Ser Val Glu Asp Cys His Tyr(SEQ ID NO:1) glycosylated form.
In the present context, the term "functionally equivalent" is intended to indicate that the homologous polypeptide has a biological activity (e.g. spasmolytic effect) corresponding to that of native HSP. The term "homologue" is intended to indicate a polypeptide encoded by DNA which hybridizes to the same probe as the DNA coding for HSP under conditions of high or low stringency (e.g. as described in Sambrook et. al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). More specifically, the term is intended to refer to a DNA sequence which is at least 60% homologous to the sequence encoding HSP with the amino acid sequence shown above. The term is intended to include modifications of the DNA sequence such as nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a mutant polypeptide with different properties than the native enzyme. Other examples of possible modifications are insertion of one or more codons into the sequence, addition of one or more codons at either end of the sequence, or deletion of one or more codons at either end or within the sequence. The term "glycosylated" is intended to indicate that a carbohydrat
REFERENCES:
patent: 4370317 (1983-01-01), Jorgensen et al.
Playford et al. Human spesmolytic polypeptide is a cytoprotective agent that stimulates cell migration. Gastroenterology. vol. 199, pp. 108-116, 1995.
Abstract -Thim et al. Biochim Biophys Acta (Netherlands) 827:(3) pp. 410-418 (1985).
Tomasetto et al., The EMBO Journal vol. 9, No. 2 pp. 407-414 (1990).
Bowie et al. Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science. vol. 247, pp. 1306-1310, Mar. 16, 1990.
Carr. H NMR-based determination of the secondary structure of porcine pancreatic spasmolytic polypeptide: one of a new family of "trefoil" motif containing cell growth factors. Biochemistry. vol. 31, No. 7, pp. 1998-2004, 1992.
Hitzeman et al. Use of heterologous and homologous signal sequences for secretion of heterologous proteins from yeast. In: Methods in Enzymology. Academic Press, Inc., NY. vol. 185, pp. 421-440, 1990.
Hoffman. A new repetitive protein from Xenopus laevis skin highly homologous to pancreatic spasmolytic polypeptide. The Journal of Biological Chemistry. vol. 263, No. 16, pp. 7686-7690, Jun. 5, 1988.
Jorgensen et al. Pancreatic spasmolytic polypeptide: I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas. Regulatory Peptides. vol. 3, pp. 207-219, 1982.
Bj.o slashed.rn S.o slashed.ren Erik
Christensen Mogens
Nielsen Per Franklin
Norris Fanny
Norris Kjeld
Gregg, Esq. Valeta A.
Kemmerer Elizabeth C.
Lathrop Brian
Novo Nordisk A S
Zelson Esq. Steve T.
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