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  Human spasmolytic polypeptide in glycosylated formUSPTO Application #: 20060019881Title: Human spasmolytic polypeptide in glycosylated form Abstract: or a functionally equivalent homologue thereof, characterized by being in glycosylated form. Glu Asp Cys His Tyr Ile Phe Glu Val Pro Trp Cys Phe Phe Pro Asn Ser Val Ser Arg Lys Cys Cys Phe Ser Asn Phe Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu Cys Ala Cys Val Met Glu Val Ser Asp Arg Arg Trp Cys Phe His Pro Leu Pro Lys Gln Glu Ser Asp Gln Phe Asp Ser Ser Val Thr Gly Val Pro Gly Ile Thr Ser Asp Gln Cys Phe Asp Asn Gly Cys Cys His Asn Arg Thr Asn Cys Gly Phe Pro Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu Ser Pro Human spasmolytic polypeptide (HSP) which has the amino acid sequence (end of abstract) Agent: Novo Nordisk, Inc. Patent Department - Princeton, NJ, US Inventors: Lars Thim, Kjeld Norris, Fanny Norris, Soren Erik Bjorn, Mogens Christensen, Per Franklin Nielsen USPTO Applicaton #: 20060019881 - Class: 514008000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Glycoprotein (carbohydrate Containing) The Patent Description & Claims data below is from USPTO Patent Application 20060019881. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 11/139,749 filed May 27, 2005, which is a continuation of U.S. Ser. No. 09/528,644 filed Mar. 20, 2000, which is a divisional of U.S. Ser. No. 09/027,893 filed Feb. 23, 1998 which is a continuation of U.S. Ser. No. 08/491,976 filed Aug. 2, 1995, now U.S. Pat. No. 5,783,416, which is a continuation of PCT/DK94/00037 filed Jan. 20, 1994, and claims priority under 35 U.S.C. 119 of Danish application serial no. 0068/93 filed Jan. 21, 1993, which is incorporated herein by reference. FIELD OF INVENTION [0002] 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 [0003] Human spasmolytic polypeptide (HSP) belongs to a family of peptides containing one or more characteristic trefoil domains [1]. The trefoil 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 forming a characteristic trefoil structure [1]. The trefoil family of peptides consists of rat intestinal trefoil factor, ITF [2], human breast cancer associated peptide, pS2 [3,4,5], porcine, human and murine spasmolytic polypeptide (PSP, HSP, MSP) [6,7,8] and frog spasmolysins (xP1, xP2 and xP4) [8,10,11] all containing 1, 2 or4 trefoil domains (FIG. 1). [0004] 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 pancreozymin or secretin [12,13,14]. PSP is resistant to digestion by intestinal proteases in the gastrointestinal tract [12], and specific binding of PSP to rat intestinal mucosa cells and membrane preparations from these cells has been demonstrated [15,16]. In the porcine gastrointestinal tract, specific receptor-like binding to Paneth cells in the duodenum has been found [17]. These results suggest a unique intraluminal function of the peptide. A pharmacological screening has indicated that PSP has spasmolytic and gastric acid secretion inhibitory effects [18], and studies on mammalian cells have indicated a growth factor-like activity of PSP [19]. [0005] The DNA sequence and derived amino acid sequence of the human counterpart of porcine SP is shown in [8]. Unlike PSP, human SP (FIG. 2), has been found to be expressed in the stomach, but not in the pancreas to any greater extent [8]. An increased expression of HSP and pS2 has been reported to be associated with peptic ulcers and mucosal injury in inflammatory bowel disease [20,21 ] indicating a possible healing function of these peptides. [0006] 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 [0007] 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. [0008] Accordingly, the present invention relates to human spasmolytic polypeptide (HSP) which has the amino acid sequence TABLE-US-00002 Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu Ser Pro His Asn Arg Thr Asn Cys Gly Phe Pro (SEQ ID NO:1) 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 or a functionally equivalent homologue thereof, characterized by being in glycosylated form. [0009] 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 comditions 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 polupeptide 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 carbohydrate moiety is present at one or more sites of the protein molecule. [0010] It is at present contemplated that glycosylation of HSP may give rise to differences in the biological activity of the protein, for instance with respect to stability towards proteolytic enzymes in the gastrointestinal tract, solubility at gastric and/or intestinal pH compared to non-glycosylated HSP, antigenicity, half-life, tertiary structure, and targeting to receptors on appropriate cells. [0011] In another aspect, the present invention relates to a variant of a spasmolytic polypeptide (SP) which is a fragment of human spasmolytic polypeptide (HSP) or porcine spasmolytic polypeptide (PSP) comprising at least one trefoil domain. [0012] The variant SP may be provided in both glycosylated and non-glycosylated form. It is at present contemplated that such a variant may be advantageous to use instead of full-length SP because of a higher specific biological activity, increased solubility and stability, longer half-life, easier way of production, or the like. [0013] It is assumed that other spasmolytic polypeptides than HSP will, if provided with a glycosylation site, also be expressed in predominantly glycosylated form. In a further aspect, the present invention therefore relates to a method of preparing a spasmolytic polypeptide in at least 60% glycosylated form, wherein a host cell transformed with a DNA fragment encoding a spasmolytic polypeptide and capable of providing glycosylation of said spasmolytic polypeptide is cultured under conditions permitting production of said spasmolytic polypeptide and recovering the resulting spasmolytic polypeptide from the culture. DETAILED DESCRIPTION OF THE INVENTION [0014] It has been found that, at least when recombinant HSP is produced in yeast, the proportion of it that is provided in glycosylated form is in N-glycosylated form. It has further been found that glycosylation takes place at Asn15 of the sequence shown above. In preferred embodiments of glycosylated HSP, the glycosylated side chain contains at least one hexose unit. In particular, the glycosylated side chain may contain at least one mannose unit, preferably at least five mannose units, most preferably at least ten mannose units. In one preferred embodiment of glycosylated HSP of the invention, the glycosylated side chain contains 13-17 mannose units. In other preferred embodiments, the glycosylated HSP is in addition glycosylated with at least one unit of N-acetyl glucosamine (GlcNAc). In the currently preferred embodiment, the glycosylated HSP is glycosylated at Asn15 with (GlcNAc).sub.2(Man).sub.10- -15. [0015] It is further contemplated to produce homologues of HSP which are provided with one or more additional glycosylation sites. Thus, the present invention also relates to HSP homologues, wherein Lys2 is replaced by Asn, Gln7 is replaced by Asn, Arg10 is replaced by Asn, Gly 20 is replaced by Thr or Ser, Gly23 is replaced by Asn, Ile 24 is replaced by Asn, Phe 36 is replaced by Asn, Asp 37 is replaced by Asn, Ser39 is replaced by Asn, Gln53 is replaced by Asn, Glu61 is replaced by Asn, Asp64 is replaced by Asn, Arg66 is replaced by Thr or Ser, Gly69 is replaced by Thr or Ser, Gly72 is replaced by Asn, Ile 89 is replaced by Thr or Ser, Pro98 is replaced by Asn or Val101 is replaced by Thr or Ser, or a combination of two or more of these substitutions. In a currently preferred embodiment of such an HSP homologue, Asp64 is replaced by Asn, and Arg66 is replaced by Thr or Ser. [0016] It is of course understood that HSP homologues of the invention may be glycosylated in the same manner at one or more of these sites as described above for glycosylation at Asn15. [0017] It is assumed that the trefoil structure common among spasmolytic polypeptides is important for the function of HSP and PSP. The variant human or porcine SP comprising a fragment of the full-length polypeptide should therefore include at least three disulfide bonds to provide this structure. Consequently, the variant may comprise at least a sequence of amino acids from position 8 to 46 or from position 58 to 95, each of which sequences defines a trefoil domain of HSP and PSP. [0018] As indicated above, the SP variant of the invention may be provided in non-glycosylated form. This may, for instance, be accomplished by substituting Asn15 by another amino acid, e.g. Asp or Glu, or by substituting Thr17 by another amino acid except Ser, e.g. Ala. It is more likely, however, that one or more additional glycosylation sites will be introduced into this domain, for instance by replacing Arg10 by Asn, Gly 20 by Thr or Ser, Gly23 by Asn, Ile 24 by Asn, Phe 36 by Asn, Asp 37 by Asn, or Ser39 by Asn, or a combination of two or more of these substitutions. Continue reading... Full patent description for Human spasmolytic polypeptide in glycosylated form Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Human spasmolytic polypeptide in glycosylated form patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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