Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-06-09
2003-07-22
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S069700, C435S252300, C435S320100, C435S471000, C536S023200, C536S023400
Reexamination Certificate
active
06596526
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to new furin polypeptides. Furin, also called PACE (for paired basic amino acid cleavage enzyme), belongs to the family of mammalian subtilisin-like proprotein convertases (SPC or PC) These proteins have been implicated in the endoproteolytic maturation processing of inactive precursor proteins at single, paired or multiple basic consensus sites within the secretory pathway (reviewed in Nakayama, 1997, Biochem.J., 327, pp. 625-635; (Seidah and Chretien, Current Opinions in Biotechnology,8, 1997, pp. 602-607). Seven distinct members of this family have been identified to date, including furin, PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6), PC7 (or LPC, PC8, or SPC7), each of which exhibits unique tissue distribution, although overlapping functional redundancy of various PCs in some tissues may occur (Seidah et al., Biochem.,1994, 76, pp. 197-209).
Furin is ubiquitously expressed in all mammalian tissues and cell lines which have been examined, and is capable of processing a wide range of bioactive precursor proteins in the secretory pathway, including growth factors, hormones, plasma proteins, receptors, viral envelope glycoproteins and bacterial toxins. It is a calcium-dependent serine endoprotease structurally arranged into several domains, namely a signal peptide, propeptide, catalytic domain, middle domain, (also termed homo-B or P-domain), the C-terminally located cysteine-rich domain, transmembrane domain and the cytoplasmic tail. Upon transit of the newly synthesized furin precursor from the endoplasmic reticulum to the Golgi compartment, the propeptide is autocatalytically removed in a two step processing event (Leduc et al., J.Biol.Chem., 267, 1992, pp. 14304-14308; Anderson et al., EMBO J., 1997, pp. 1508-1518).
Furin is predominantly localized to the trans-Golgi network (TGN), but it also cycles between the TGN and the cell surface via endosomal vesicles, thereby processing both precursor proteins during their transport through the constitutive secretory pathway as well as molecules entering the endocytic pathway. The cellular distribution of furin to the varied processing compartments is apparently directed by defined structural features within its cytoplasmic tail (Schäfer et al., EMBO J.,11, 1995, pp. 2424-2435; Voorhees et al., EMBO J., 20, 1995, pp. 4961-4975; Teuchert et al., J.Biol.Chem., 274, 1999, pp. 8199-8207). Deletion of the cytoplasmic domain results in a truncated furin polypeptide located primarily in the plasma membrane, to which it is transported probably by a default pathway, incapable of recycling to the TGN due to the loss of regulative sequence motifs within the cytoplasmic domain (Molloy et al., EMBO J., 13, 1994, pp. 18-33; Schäfer et al., EMBO J., 14, 1995, pp. 2424-2435).
The C-terminal domains have been found to be dispensable for the functional activity of furin. Mutant furin lacking the transmembrane domain and the cytoplasmic tail, was found to be readily released into cell culture medium while still exhibiting significant activity. High levels of expression of full length recombinant furin have resulted in the natural secretion of a truncated furin form, called ‘shed’ furin, which lacks the transmembrane domain and the cytoplasmic tail (Wise et al., Proc.Natl.Acad.Sci., 87, 1990, pp. 9378-9382; Rehemtulla and Kaufman, Blood, 79, 1992, pp. 2349-2355; Vidricaire et al., Biochem.Biophys.Res.Comm., 195, 1993 pp. 1011-1018; Vey et al., J.Cell.Biol., 127, 1994, pp. 1829-1842; Preininger et al.,Cytotechnol., 30, 1999, pp. 1-15). It remains an open question as to whether furin shedding is due to saturating cellular retrieval mechanisms, whether it represents a protection mechanism of the host cell against excess protease, or whether is part of a natural regulatory process modulating intracellular furin concentration/activity by secretion. The isolation of a truncated endogenous furin from the Golgi fraction of bovine kidney cells may support the view that shedding is not solely an artificial secretion process caused by overexpression (Vey et al., 1994). Conversion of furin into the soluble secreted form was shown to occur intracellularly within an acidic compartment which requires the presence of calcium (Vey et al., 1994).
The presence of a C-terminal truncated and hence soluble form of furin that remains active, however, has been detected almost exclusively in conditioned medium of cells recombinantly overexpressing native full-length furin (Wise et al., 1990; Rehemtulla and Kaufman, 1992; Vidricaire et al., 1993; Vey et al., 1994; Preininger et al., 1999).
Other prior art describing furin polypeptides includes WO 91/06314, which describes a fragment of furin consisting of amino acids 108-464, thus lacking part of the homo-B domain, the cysteine-rich region, the transmembrane domain and the cytoplasmic tail. WO 92/09698 discloses full length furin and furin lacking the transmembrane domain. In addition, Preininger et al. (Cytotechnology 30, 1999, pp. 1-15) describe furin mutants lacking the cysteine rich region, the trans-membrane domain and the cytosolic domain. Cells expressing such mutants contained increased intracellular concentrations of the furin derivatives but varying levels of secretion. The authors stated that the lack of extracellular accumulation of these molecules suggested that these molecules were most likely degraded. The authors stated further that full length recombinant furin, located intracellularly, seems to be largely inactive and that there is a potential toxicity of larger amounts of full length furin to its host cell.
SUMMARY OF THE INVENTION
We have found that soluble furin in a cell culture medium can cause proteins which are not naturally processed by furin to be unspecifically cleaved. For example, although native Factor VIII is not naturally processed by furin, Factor VIII can become a target for inadvertant processing by soluble furin when exposed to furin for an extended period of time, e.g. in a cell culture medium. This leads to, a reduced yield of structurally intact Factor VIII protein in such cell culture medium. This can be the case when Factor VIII is coexpressed together with a natural substrate of furin, e.g. von Willebrand Factor, or when recombinant proteins which are naturally processed by furin are exposed to furin for an extended period of time so that in addition inadvertent sites are cleaved.
The present invention reduces or prevents unspecific cleavage of proteins in cell culture through the use of modified furin polypeptides which have proteolytic activity but which are not secreted into culture medium by host cells or are secreted in reduced amounts compared to the secretion of wild-type furin. Such furin polypeptides have been found not to be toxic to host cells even when expressed intracellularly in high amounts.
Accordingly, the present invention provides a furin polypeptide having a modified amino acid sequence compared to that of wild-type furin between homo-B-domain and the transmembrane domain, that is, between amino acids Ala 557 and Leu 713 according to the amino acid sequence presented in
FIGS. 1 and 2
. It has been surprisingly found that furin polypeptides having such a modified amino acid sequence have proteolytic activity similar to that of native (i.e., wild-type) furin, but are secreted by host cells expressing such furin polypeptides into cultivation medium in substantially reduced amounts compared to native furin.
It is another aspect of the invention that the furin polypeptides according to the invention can be expressed in high amounts in a cell without being substantially toxic to the cell. In still a further aspect, the physiological cleavage properties of the modified furin protein are still present, but inadvertent cleavage of secreted or extracellularly localized proteins in a cell culture medium is highly reduced since less or no furin is present in the medium.
Additionally, a further advantage of the furin polypeptide of the present invention is that although the proteolytic processing
Plaimauer Barbara
Schlokat Uwe
Baxter Aktiengesellschaft
Fedrick Michael F.
Moore William W.
Nashed Nashaat T.
Townsend and Townsend and Crew
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