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
1993-03-01
2002-02-19
Kunz, Gary L. (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...
C435S320100, C435S325000
Reexamination Certificate
active
06348327
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to host cells expressing one or more prohormone convertase enzymes and the production of a biologically active polypeptide from these cells. The invention further relates to polypeptide precursor variants having endoprotease cleavage sites which are processed by the host cell.
2. Description of Related Art
Most, if not all proteinaceous hormones are synthesized as relatively large precursor molecules, prohormones, that are biologically inactive (reviewed in Docherty and Steiner 1982; Loh et al. 1984; Mains et al. 1980). Maturation of the prohormone to its active form often requires endoproteolytic cleavage at paired or multiple basic amino acid residues to liberate the active component from the inactive portion of the precursor molecule. Until recently, almost nothing was known concerning the identity of the proteins responsible for this important stage in processing: the prohormone convertase (PC) enzymes.
Not every kind of cell has the capacity to correctly convert a prohormone to its active mature form through these specific cleavages. For some classes of prohormone this processing is apparently limited to those cells that contain both constitutive and regulated pathways of protein secretion (Gumbiner and Kelly 1982). Cells having both constitutive and regulated pathways of protein secretion are located almost exclusively in the specialized hormone-producing tissues of the endocrine and neuroendocrine systems.
An example of a family of hormones that is processed during regulated secretion is the insulin family of hormones. This family includes insulin, the insulin-like growth factors IGF-I and IGF-II, and relaxin. In their native environment, all members of this family are synthesized as precursor molecules that require processing to yield active hormone.
When heterologously expressed in cells having only a constitutive pathway of protein secretion, most hormone precursors, such as human preproinsulin, are secreted in an unprocessed prohormone form (Gumbiner and Kelly 1982). Experimental manipulation of mouse AtT-20 cells that disrupted the regulated secretory pathway of those cells has been observed to redirect the polyhormone precursor proopiomelanocortin (POMC) into the constitutive secretory pathway. In that case, POMC was no longer subjected to processing and was found to be secreted from the cell as the intact precursor (Moore et al. 1983). These observations suggest that there is a class of processing enzymes that function only in the regulated pathway of protein secretion; this pathway is apparently limited to certain highly-specialized cell types.
The POMC protein is a prohormone that is subject to differential processing. Expression of mature POMC derivatives is highly tissue-specific; alternate processed forms of the same prohormone precursor are produced in different regions of the brain (Douglass et al. 1984). The enzyme(s) and control mechanisms involved in the generation of this diversity are unknown. The possibility exists that there are tissue-specific enzymes that recognize unique amino acid sites on the prohormone substrate, or alternatively, that only one enzyme is responsible for the endoproteolytic cleavage and is itself under tight metabolic control, with each tissue providing a characteristic intracellular environment that is associated with cleavage at a specific subset of residue pairs.
Until recently, the only known eukaryotic prohormone processing enzyme was the KEX2 gene product of the yeast
Saccharomyces cerevisiae
(Jullius et al. 1983 and 1984; Fuller et al. 1989a.) The kex2 protein is a serine protease related to the subtilisin family of enzymes and has a preference for specific pairs of basic amino acids on its native hormone precursor substrates (pro-a-factor mating type pheromone and the pro-killer toxin). Kex2 shows maximum enzymatic activity at neutral pH with a strict requirement for the presence of calcium (Julius et al. 1984; Fuller et al. 1989a). It is membrane-bound and the mature, active form of the enzyme is localized in the post-Golgi compartment of the yeast cell (Fuller et al. 1989b; Redding et al. 1991). It can effectively serve as a substitute convertase for bona fide mammalian PC enzymes when heterologously expressed in otherwise processing-deficient cells by its demonstrated ability to correctly process certain mammalian prohormones: Nerve growth factor, bNGF, in BSC-40 cells (Bresnahan et al. 1990); protein C in baby hamster kidney BHK cells (Foster et al. 1991); POMC both in BSC-40 cells (Thomas et al. 1988) and in COS-1 cells (Zollinger et al. 1990)]. Kex2 was shown to have a highly similar if not identical substrate specificity to the authentic human proalbumin convertase in vitro (Bathurst et al. 1986; Brennan et al. 1990). When heterologously expressed in mammalian cells kex2 will home to the post-Golgi compartment, where it is apparently fully active (Germain, et al. 1990). These observations have led to speculation that this yeast protein must be both functionally and structurally similar to an authentic mammalian convertase. A search began for the elusive mammalian counterparts of kex2 based upon structural homologies.
KEX2 and Fur Hydrophobic Anchor
The Kex2 endoprotease has two hydrophobic regions located at the N-terminal side and C-terminal side. The C-terminal hydrophobic transmembrane anchor is responsible for the anchoring of the Kex2 to a Golgi body of a yeast cell. Deletion of this C-terminal hydrophobic anchor renders the Kex2 endoprotease soluble while still maintaining substrate specificity (EPO PUB No.0327377).
An inspection of genetic data bases identified a potential mammalian homologue of kex2 that shared many features of the active site domain of the kex2 protein: the fur gene product of human liver, furin (Fuller et al. 1989b). Furin was subsequently cloned and successfully expressed in processing-deficient cells: cotransfection of furin with pro-von Willebrand factor in COS-1 cells (van den Ven et al. 1990; Wise et al. 1990) and with pro-bNGF in African green monkey kidney epithelial BSC-40 cells (Bresnahan et al. 1990) resulted in correct processing of the precursor substrates.
However, when furin and a prohormone, prorenin, have been coexpressed in mammalian cells, no processing has been observed (Hatsuzawa, et al
The Journal of Biological Chemistry
Vol 265 [1990]).
Furin also shared with kex2 a requirement for calcium ions, displayed maximum activity at a neutral pH, and, like kex2, was shown to be a membrane-bound protein in the post-Golgi compartment (Bresnahan et al. 1990). However, because furin does not seem to be capable of efficiently processing certain hormone precursors such as prorenin, and its mRNA message is apparently expressed in most if not all mammalian cells (Hatsuzawa et al. 1990), the furin protease may play a role in an essential “housekeeping” function in that it could be responsible for many of the basic amino acid site cleavages occurring in the constitutive secretory pathway of the cell. These functions could be general, or more confined to specific cell-types with the constitutive pathway-dependent processing of growth factors like bNGF.
Because furin does not appear to be directly involved in the endoproteolytic processing of prohormones in endocrine-like tissues, there must be other mammalian proteins both functionally and structurally similar to furin and yeast kex2 that serve as the authentic prohormone convertases: PC proteins that share distinctive homologies in their active sites. This search for structural homology has recently lead to the discovery of more PC proteins.
By using the technique of “Mixed Oligonucleotides Primed Amplification of cDNA” (MOPAC, Innis 1990), Smeekens and Steiner (1990) were able to use the conservation of amino acid sequence surrounding the active sites of both bacterial subtilisin and the yeast kex2 protease to amplify a putative prohormone convertase cDNA from human insulinoma. This PC cDNA, termed mPC2, showed an exceptional degree of
Gorman Cornelia M.
Groskreutz Debyra J.
Genentech Inc.
Gucker Stephen
Kunz Gary L.
Merchant & Gould P.C.
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