Stabilization of cardiac troponin I subunits and complexes

Details Stabilization of cardiac troponin I subunits and complexes Stabilization of cardiac troponin I subunits and complexes

2001-04-27

2003-03-25

Weber, Jon P. (Department: 1651)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C424S193100, C424S194100, C435S188000, C435S302100, C435S302100, C530S350000

Reexamination Certificate

active

06538104

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to the conjugation of individual and complexed subunits of multisubunit protein complexes with polymers to stabilize their conformation. More specifically, the present invention describes a method of stabilizing individual and complexed subunits via covalent conjugation to a natural or synthetic polymer. The present invention also relates to stabilized conjugates of cardiac troponin I and C, and methods for its preparation.
SUMMARY OF THE INVENTION
Proteins are composed of long chains of amino acids. The structure of proteins can be considered on four different levels. The primary structure refers to the specific order of amino acids in the polymer chain. The secondary structure refers to the interactions among and between the amino acids in the chain to form such structures as &agr;-helices and &bgr;-pleated sheets. The tertiary structure refers to the three-dimensional structure of the protein, which is also referred to as a protein's conformation. The quaternary structure refers to the spatial arrangement of individual polypeptides or subunits of multisubunit proteins.
The native conformation of a protein is only marginally stable. Thus, many proteins which are removed from their native environment and purified undergo conformational changes which can cause a loss of biological activity, such as enzyme activity or antibody binding capacity. In particular, the individual (uncomplexed) subunits of multisubunit protein complexes may undergo dramatic conformational changes when separated from the other subunits of the complex and stored in a liquid medium.
It is often desirable to separate the individual subunits of a multisubunit protein complex, for example to study or exploit the biological activity of each individual subunit. However, this may not be possible if the individual subunits undergo conformational changes in their uncomplexed state that alter their biological activity. Therefore, it is often desirable to stabilize specific subunit complexes of more than one, but less than all, subunits of a multisubunit protein. Accordingly, it is an object of the present invention to provide a method for stabilizing specific subunit complexes of multisubunit proteins.
Troponin is an example of a multisubunit protein complex which consists of three individual subunits; troponin T, troponin C, and troponin I. The troponin complex is involved in the calcium-sensitive switch that regulates the interaction of actin and myosin in striated muscles. Troponin T binds the troponin complex to tropomyosin, while troponin I is the inhibitory subunit of the complex, because it inhibits the actomyosin Mg
2+
-ATPase. Whereas troponin (TnC), which binds Ca
2+
, from skeletal muscle and cardiac muscle is identical, troponin I and T (TnI and TnT) from these two sources exist as different isoforms, each having a different amino acid sequences and thus a unique structure. Thus, cardiac troponin I (cTnI), cardiac troponin T (cTnT), and subunit complexes, such as cardiac troponin I and C (cTnIC) are of particular interest as cardiospecific markers.
The majority of the research into the troponin complex has centered around the regulatory function and structure of the troponin complex in skeletal muscle. The troponin complex assists in muscle contraction. The TnC molecule has four binding domains to bind divalent metal ions. The Ca
2+
/Mg
2+
binding sites are in the C-terminal region and the Ca
2+
binding sites are in the N-terminal region. In studies of skeletal muscle, in the absence of Ca
2+
, the N-terminus of TnI binds to the C-terminal region of TnC and to the globular C-terminal region of TnT. Thus, research indicates that TnI and TnC are anti-parallel and TnI and TnT are anti-parallel. The presence of calcium ion increases the C-terminal domain's affinity for the inhibitory and C-terminal regions of TnI. In addition, there is a hydrophobic surface in the N-terminal domain of TnC that represents a Ca
2+
dependent binding site for TnI and TnT. It has been proposed that the Ca
2+
dependent reactions relate to the regulatory mechanism and Ca
2+
independent interactions maintain the structural integrity of the complex. In order to study structure and function of the troponin complex in its regulation of skeletal muscle, cross-linking studies have been accomplished. See Farah, C. and Reinach, F. Review: The Troponin complex and regulation of muscle contraction. FASEB Journal 9 pp. 755-767 (1995). Covalent binding between TnC and skeletal muscle TnI has been formed between the carboxyl groups in the TnC and lysine groups in TnI using EDC. See Kobayoshi et al. (1994), Structure of the troponin complex: implications of photocross-linking of troponin I to troponin C thiol mutants. J. Biol. Chem. 269, 5725-5729. In addition, Leszyk et al. (1987) Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-malemidobenzophenone; identification of residues in troponin I that are close to cystein-98 of troponin C.
Biochemistry
26, 7042-7047, reported that the main product of cross-linking between TnC and skeletal muscle TnI comprises segments derived from the N-terminal regulatory domain of TnC (residues 46 to 78) and the inhibitory region of skeletal TnI (residues 9-116).
U.S. patent application Ser. No. 08/865,468, filed on May 29, 1997, discloses that the majority of native cTnI in human serum after myocardial infarction (MI) is associated with TnC and TnT. The presence of TnI in a complex with other troponin subunits in MI patient serum increases its stability and protects it from further degradation. In addition, the troponin complex protects the sites where cardiac-specific antibodies bind. U.S. patent application Ser. No. 08/865,468, filed on May 29, 1997, also discloses methods to isolate the complex from MI patient serum.
The determination of the presence or amount of certain constituents or analytes is useful in the diagnosis of disease and physical well-being. Compositions which behave similarly to how constituents present in human bodily fluids behave, e.g., blood, blood serum, plasma, spinal fluid, and urine, are used in clinical laboratories. These compositions assist in the determination of whether the clinical instrumentation and procedures used by the laboratory to measure the constituents are accurate. These compositions are also used to calibrate the clinical devices which measure the amount or presence of the constituent in a sample. These compositions will be referred to hereinafter as control compositions or controls.
Rapid and simple tests that can be used to accurately diagnose the occurrence of myocardial infarction or distinguish other ischemic events such as unstable angina are extremely important. Cardiac troponin I (cTnI) and troponin T (cTnT) have recently become established as the markers of choice in evaluating cardiac distress. See for example, New England Journal of Medicine Volume 335 No. 18, pages 1342-1349, Antman et al. and pages 1333-1341, Ohman et al.
A variety of immunoassays have been developed that utilize antibodies that can distinguish between the three troponin subunits, and also between their different isoforms. Monoclonal and polyclonal antibodies have been designed and used in immunoassays which can detect the cardiac-specific epitopes formed by the unique amino acid sequence of cTnI. See for example, International Patent Application No. WO 96/10076; European Patent No. 394,819 B1; and Adams et al., Circulation 88:101-106 (1993). Larue et al., (Clin. Chem. 39:972-979 (1993)) describe an immunoenzyme assay that is capable of detecting cTnI in the concentration range of 0.2 to 20 &mgr;g/L in 30 minutes.
Immunoassays have also been described which are specific for TnT. See for example, Katus, et al., Circulation, 83(3):902-912 (1991). An immunoassay for TnT is also commercially available from Boehringer Mannheim Corporation (Indianapolis, Ind.).
Most immunoassays are designed to determine the concentration of a given marker in

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