Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-08-21
2001-05-15
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S219000, C435S815000
Reexamination Certificate
active
06232089
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to processes for identifying and characterizing a novel enzyme. This invention relates to the characterization of a protein expressed on the surface of a variety of cells. The protein is of importance in the human immune response and regulation of IgE production.
BACKGROUND OF THE INVENTION
CD23 (the low affinity IgE receptor, FceRII) is a Type II 45 kDa protein expressed on the surface of various cells, especially mature B-cells, monocytes and macrophages (Delespesse, et al, Adv. Immunol. 49, 149-191 (1991); Gordon, Immun. Today 15, 412-417 (1994)). The structure of CD23, based on its predicted amino acid sequence and homology to other proteins, consists of an N-terminal cytoplasmic domain, a single transmembrane helix, a stalk region and a lectin domain (Ikuta et al, Proc. Nat'l. Acad. Sci. USA 84, 819-823 (1987), Beavil et al, Proc. Nat'l. Acad. Sci. USA 89, 753-757 (1992)). The stalk region is predicted to form a leucine zipper, resulting in oligomerization of CD23 on the cell surface. CD23 expressed on the cell surface is cleaved to soluble forms (sCD23), first identified as IgE binding factors, as these CD23 fragments retain the ability to bind IgE via the lectin domain. The cleavages are known to occur in the stalk region based on the N-terminal sequence of the purified soluble forms of CD23. The two largest fragments begin, respectively, at amino acids 81 and 102 of the human CD23 sequence and are identified as the 37 kDa and 33 kDa soluble CD23 fragments based on their molecular weight as determined by denaturing polyacrylamide electrophoresis; an additional fragment has also been identified as beginning at amino acid 125 with an apparent molecular weight of 29 kDa which is a minor cleavage product (Letellier et al, Molec. Immun. 26, 1105-1112 (1989)).
Additional fragments of CD23 have also been described of apparent molecular weight 25-27 kDa (Bonnefoy et al., Eur. J. Immun. 18, 117-122 (1988); Sarfati et al, Immunology 60, 539-545 (1987)). The 25 kDa fragments correspond to the major form of IgE binding factors found circulating in human serum (Bujanowski-Weber et al., Immunology, 65, 53-58 (1988); Yukawa et al., J. Immun., 8, 2576-2580 (1987)). These smaller fragments are proposed to be derived from the 37, 33 and 29 kDa fragments by an autocatalytic mechanism different from that which produces the larger fragments (Letellier 35 et al, J. Exp. Med. 172, 693-700 (1990)), although this remains unproven. Addition of 10-20 mM iodoacetamide to cell cultures prevents the accumulation of the 25-27 kDa fragments, but results in the accumulation of 37 and 33 kDa fragments (Letellier et al., J. Immun. 141, 2374-2381 (1988)). Cleavage of CD23 to the larger fragments would then be necessary but not sufficient for production of the 25-27 kDa soluble CD23 fragments. Several reports (Bonnefoy et al., Eur J Immun 18, 117-122 (1988);Sarfati et al, Immunology 60, 539-545 (1987); Moulder et al Eur. J. Immun. 23, 2066-2071 (1993); Bujanowski-Weber et al., Immunology 65, 53-58 (1988)) indicate that under some conditions the 25 kDa fragments accumulate very rapidly in the culture supernatant of CD23-expressing cells, with very little of the larger fragments observed. The cleavage of CD23 from the 37, 33, 29 kDa fragments to the 25-27 kDa fragments may therefore be catalyzed by a different process than that giving rise to the 37, 33 and 29 kDa fragments. The properties of the enzyme responsible for the release of the large soluble CD23 fragments, i.e. the CD23 processing enzyme, have not yet been described.
CD23 has been implicated in human immune response, most clearly in the regulation of IgE production through binding of IgE to CD23 as the low affinity IgE receptor and by immunostimulation via the cytokine activity of the soluble fragments. Particular activities of intact cell-bound CD23 include: a) antigen presentation, b) IgE mediated eosinophil cytotoxicity, c) B cell homing to germinal centres of lymph nodes and spleen, and d) down regulation of IgE synthesis (Delespesse et al, Adv Immunol, 49, 149-191 (1991)). The soluble CD23 fragments (apparent molecular weight 37, 33, 29 and 25 kDa) have multifunctional cytokine properties which appear to play a major role in IgE production. Thus, the excessive formation of soluble CD23 fragments has been implicated in the overproduction of IgE, the hallmark of allergic diseases such as extrinsic asthma, rhinitis, allergic conjunctivitis, eczema, atopic dermatitis and anaphylaxis (Sutton and Gould, Nature, 366, 421428 (1993)). Other biological activities attributed to soluble CD23 fragments include the stimulation of B cell growth and a variety of proinflammatory processes such as the induction of the release of cytokines from monocytes mediated by binding of sCD23 to a receptor (Lecoanet-Henchoz, et al., Immunity 3, 119-125 (1995)). Elevated levels of soluble CD23 have been observed in the serum of patients having B-chronic lymphocytic leukaemia (Sarfati et al, Blood, 71, 94-98 (1988)) and in the synovial fluids of patients with rheumatoid arthritis (Chomarat et al, Arthritis and Rheumatism, 36, 234-242 (1993); Plater-Zyberk and Bonnefoy, Nature Med 1, 781-785 (1995)).
The expression of a CD23 processing enzyme is expected in the many cells which express CD23 in the numerous roles identified for CD23 and the soluble fragments. There exists a need to characterize and purify the CD23 processing enzyme as its importance in the immunologic field is clearly recognised and modulation of its activity will have therapeutic utility.
SUMMARY OF THE INVENTION
The present invention is to the discovery of a novel CD23 processing enzyme preparation. A CD23 processing enzyme preparation, which is a membrane bound preparation, has been found to have the following characteristics:
a) an enzyme activity which produces MW, app 37 and 33 kDa CD23 fragments in the membrane cleavage assay (as described herein);
b) the activity is inhibited by [4-(N-Hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)-succinyl]-(S)-phenylalanine-N-methylamide and 1,10-phenanthroline; and
c) the activity is not inhibited by the protease inhibitors E-64, PMSF, leupeptin, pepstatin, and TLCK in the membrane assay.
Suitably the preparation also has a pH for optimum activity which is above pH 7.5, and continues to a pH of up to about pH 9.0, and has decreasing activity at a pH below about 7.5.
The novel CD23 processing enzyme preparation may also be characterized by a solubilized preparation which has the following characteristics:
a) an enzyme activity that produces a MW, app 33 kDa CD23 fragment using the solubilized membrane assay (as described herein);
b) a pH for optimum activity which is above pH 7.5, and continues to a pH of up to about pH 9.0, and has decreasing activity at a pH below about 7.5;
c) the activity is inhibited by [4-(N-Hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)-succinyl]-(S)-phenylalanine-N-methylamide and 1,10-phenanthroline; and
d) the activity is not inhibited by the protease inhibitors E-64, PMSF, leupeptin, pepstatin, and TLCK in the membrane assay.
The solubilized preparation may also further have the following characteristics:
a) an apparent MW by gel filtration, between about 45 to about 60 kDa;
b) an activity which binds to metal chelating column;
c) binds to an affinity column made with N-[(3-(S)-propargylthiomethyl-4-(N-Hydroxyamino)-2R-isobutyl)succinyl]-(S)-phenylalanine-N-(6-biotinoyl-aminohexyl)amide and which is eluted by addition of an excess of another CD23 processing inhibitor.
Suitably the metal in the chelating column is a divalent metal, such as Zn, and the column material is preferably a Sepharose based column. Any suitable gel filtration column may be utilized herein, suitably the column is a Superose 12 column, or a Sephacryl S-300 (Pharmacia) column.
Another aspect of the present invention is to a method for preparing purified CD23 processing enzyme preparations. The first of such methods comprise:
a) preparing purified plasma membr
Buckle Derek Richard
Christie Gary
Marolewski Ariane Elizabeth
Mayer Ruth Judik
Smith David Glynn
Dinner Dara L.
Kinzig Charles M.
Meller Michael
Simon Soma G.
SmithKline Beecham Corporation
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