Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-09-19
2004-09-28
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S023000, C530S324000, C530S330000
Reexamination Certificate
active
06797461
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to assays to detect tryptase activity and polypeptide tryptase substrates utilized in the assay.
BIBLIOGRAPHY
Complete bibliographic citations to the references noted herein are included in the Bibliography section, immediately preceding the claims.
DESCRIPTION OF THE RELATED ART
Mast cells are distributed on all epithelial and mucosal surfaces of the body. In addition to being found in mucous membranes of the respiratory and gastrointestinal tract, mast cells are also located near blood vessels in connective tissue. Mast cells play an important role in innate and acquired immune responses through the release of dense granules upon activation. The major component of mast cell secretory granules is serine proteases (Schwartz, L., et al.).
Human &bgr;-tryptase is the most abundant and unique member of the serine protease family. Although &bgr;-tryptase has uncertain physiological functions, it has been implicated as an effector in a plethora of human allergic and pathophysiological conditions, including asthma, arteriosclerosis, cancer, otitis media, arthritis, interstitial cystitis, rhinitis, dermatitis, and other deep organ diseases. Its prominent role in tissue remodeling and angiogenesis is suggestive of potentially beneficial physiological processes. There are at least three proteolytically active isoformns of tryptase present in mast cells, &bgr;I-tryptase, &bgr;II-tryptase, and &bgr;III-tryptase. These tryptase isoforms are secreted as catalytically active tetramers (~135 kD) that are resistant to inactivation by plasma inhibitors.
The &bgr;-tryptase enzyme has been recently crystallized, and the structure suggests that the association of the tryptase subunits into the native tetramer results in a stereospecific admission of potential substrates to the active site of each subunit. Although several in vitro studies have identified multiple substrates for tryptase, including neuropeptides, fibrinogen, stromelysin, pro-urokinase, prothrombin, and protease activated receptor-2, the physiologically relevant in vivo target of &bgr;-tryptases's serine protease activity has eluded discovery.
Human chromosome 16 encodes at least four homologous, yet distinct, tryptase genes, designated &agr;-, &bgr;I-, &bgr;11-, and &bgr;III-tryptase (Pallaoro, M., et al.). As used herein the unmodified term “tryptase” shall be used to refer to all tryptase isoforms. Two &bgr;-tryptase isoforms share greater than 99% sequence identity, the &bgr;I- and &bgr;II-tryptases differing by only a single N-glycosylation site. It is not clear why so many highly similar tryptases are expressed by mast cells. One possibility is that they each perform different proteolytic functions that may be reflected in their substrate specificity preferences. Indeed, it has recently been shown that a single amino acid substitution between tryptase &agr; and tryptase &bgr;II accounts for discrimination in substrate preference for the two enzymes (Huang, C., et al.).
It has been difficult to study &bgr;-tryptase and its physiological role because there are no suitable animal models for human allergies. Further, the human &bgr;-tryptases show little or no homology with the tryptases found in animals other than primates. Finally, isolating natural &bgr;-tryptase from human cadavers is a tedious and biohazardous undertaking. Only recently has recombinant, enzymatically-active tryptase become available through the work of the assignee of the present application, Promega Corporation of Madison, Wis., USA. (See co-pending and co-owned U.S. patent applications Ser. No. 09/598,982, filed Jun. 21, 2000, and Ser. No. 091079,970, filed 15 Apr. 1998, the entire contents of which are incorporated herein.)
During the past decade, clinicians have appreciated and reported the value of measuring released tryptase in making atopic diagnoses as well as when monitoring the course of mast cell-mediated disease. (“Atopic” being an umbrella term designating disease states characterized by symptoms produced upon exposure to an excitatory antigen or conditions such as asthma and other allergic reaction) &bgr;-tryptase may be detected in the serum of non-atopic “normal” individuals, and population serum levels are typically less than 1000 picograms of &bgr;-tryptase per milliliter of serum. Conversely, serum tryptase levels are markedly raised in atopic subjects. Too often however, immunological detection (i.e., ELISA, RIA, PCFIA, and related assays of tryptase) is fraught with poor sensitivity or availability (e.g., the Schwartz ELISA method) and the requisite need for expensive ancillary detection equipment. Except in cases of exaggerated mast cell burden or degranulation, such as occurs during mastocytosis or anaphylaxis, it has been difficult to establish non-atopic or remission baselines of tryptase.
Conventional methods of assaying for tryptase proteolytic activity are hampered by poor specificity. These methods use substrates that are only intended for the measurement of “trypsin-like” activity, particularly in purified tryptase preparations. For example, Benzoyl-Arg-paranitroaniline (trypsin), Tosyl-Gly-Pro-Arg-pNa (thrombin), Tosyl-Gly-Pro-Lys-pNa (plasmin), and Tosyl-Arg-Methyl-Ester exhibit cleavage upon contact by tryptase, but also are cleaved by other serine proteases. Because tryptase and related blood-borne serine proteases are able to cleave these substrates, they are of little value in ascertaining tryptase activity levels in complex biological samples.
SUMMARY OF THE INVENTION
The invention, which is defined by the claims set out at the end of this disclosure, is intended to solve at least some of the problems noted above.
A first embodiment of the invention is directed to an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, wherein P4 is Proline (“P”), P3 is Arginine (“R”) or Lysine (“K”), P2 is any amino acid, and P1 is K or R (SEQ. ID. NO: 1). For amino acid abbreviations, see Table 1 below. These isolated polypeptides function as very specific substrates that can be cleaved by the action of tryptases.
A second embodiment of the invention is directed to a method of assaying activity of an enzymatically-active &bgr;-tryptase in a sample. The method comprises first contacting the sample with an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, where P4 is P, P3 is R or K, P2 is any amino acid, and &bgr;I is K or R (SEQ. ID. NO: 1). The isolated polypeptide also includes a detectable leaving group bound to P4-P3-P2-P1, and is amino-terminally blocked. The sample is contacted with the isolated polypeptide under conditions wherein an amount of the detectable leaving group is cleaved from P4-P3-P2-P1 upon action of &bgr;-tryptase present in the sample. The amount of detectable leaving group cleaved from the polypeptide is then quantified to give an indication of the extent of tryptase activity in the sample.
In the preferred embodiment of the method, the sample is contacted with an isolated polypeptide comprising in amino to carboxy order P4-P3-P2-P1, wherein P4 is acetylated, and wherein P4-P3-P2-P1 is selected from the group consisting of P-R-N-K (SEQ. ID. NO: 2), P-K-N-K (SEQ. ID. NO: 3), P-R-N-R (SEQ. ID. NO: 4), P-K-N-R (SEQ. ID. NO: 5), P-A-N-K (SEQ. ID. NO: 6), and P-R-T-K (SEQ. ID. NO: 7) (wherein asparagine is “N” and threonine is “T”), and further wherein a fluorogenic leaving group comprising 7-amino-4-carbamoylmethyl- coumarin is bound via an amide bond to P4-P3-P2-P1 at a carboxy-terminus of P4-P3-P2-P1. Here, if the sample has any tryptase activity, such activity will produce a detectable fluorescent moiety. The fluorescence of the sample is then measured to determine whether it undergoes a detectable change in fluorescence, the detectable change being an indication of the activity of the enzymatically-active &bgr;-tryptase in the sample. The sample may be any sample suspected of containing tryptase activity, including whole blood, serum, plasma, urine, tears, lavage, tissue extract, conditioned media, etc.
A third embodiment of
Craik Charles S.
Haak-Frendscho Mary
Harris Jennifer L.
Niles Andrew L.
Celsa Bennett
DeWitt Ross & Stevens S.C.
Leone, Esq. Joseph T.
Promega Corporation
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