Substrates for assessing mannan-binding protein-associated...

Details Substrates for assessing mannan-binding protein-associated... Substrates for assessing mannan-binding protein-associated...

1999-02-08

2001-05-22

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S004000, C435S968000, C435S975000, C530S300000

Reexamination Certificate

active

06235494

ABSTRACT:

FIELD OF THE INVENTION
Provided herein are assays for measuring in vivo levels of activated mannan-binding protein-associated serine protease (MASP-1 and MASP-2) activity. Also provided are compounds that are useful for assessing the in vivo levels and for monitoring in vitro and in vivo complement-activation (C-activation).
BACKGROUND OF THE INVENTION
The complement (C) system of humans and other mammals involves more than 20 components that participate in an orderly sequence of reactions resulting in complement activation. The blood complement system has a wide array of functions associated with a broad spectrum of host defense mechanisms including anti-microbial and anti-viral actions (Müller-Eberhard (1988)
Annu. Rev. Biochem.
57:321-347; Rother et al. (1984) in Contemporary Topics in Immunology, Vol. 14 (Snyderman, R., Ed.), pp. 109-153, Plenum Publishing Company, New York). Products derived from the activation of C components include non-self recognition molecules C3b, C4b and C5b, as well as the anaphylatoxins C3a, C4a and C5a that influence a variety of cellular immune responses (Hugli et al. (1982) 15th International Leucocyte Culture Conference, Asilomar, Calif. (Abstract); Fujii et al. (1993)
Protein Science
2:1301-1312; Morgan et al. (1982)
J. Exp. Med.
155:1412-1426; Morgan (1993)
Complement Today
1:56-75; Morgan et al. (1983)
J. Immunol.
130:1257-1261). These anaphylatoxins also act as pro-inflammatory agents (Chenoweth et al. (1983)
Agents Actions
12:252-273; Hugli et al. (1978) in Advances in Immunology, Dixon et al., Eds., pp. 1-53, Academic Press, New York). The role of C in The C system also has a role in immuno-pathogenesis of a number of disorders, including autoimmune diseases such as rheumatoid arthritis (see, e.g., Wang et al. (1995)
Proc. Natl. Acad. Sci. U.S.A.
92:8955-8959; Moxley et al. (1987)
Arthritis
&
Rheumatism
30:1097-1104), lupus erythematosus (Wang et al. (1996)
Proc. Natl. Acad. Sci. U.S.A.
93:8563-8568; and Buyon et al. (1992)
Arthritis Rheum.
35:1028-1037) and acute glomerulonephritis (Couser et al. (1995)
J. Am. Soc. Nephrol.
5:1888-1894). Other pathologies that involve activation of the C system include sepsis (see, e.g., Stove et al. (1996)
Clin. Diag. Lab. Immunol.
3:175-183; Hack etal. (1989)
Am. J. Med.
86:20-26), respiratory distress syndrome (see, e.g., Zilow et al. (1990)
Clin. Exp. Immunol.
79:151-157; and Stevens et al. (1986)
J. Clin. Invest.
77:1812-1816) and multiorgan failure (see, e.g., Hecke et al. (1997)
Shock
7:74; and Heideman etal. (1984)
J. Trauma
241038-1043). Interest in such pathologies as well as interest in C-activation associated with transplanted organ rejection (see, e.g., Dalmasso et al. (1992)
Immunopharmacology
24:149-160; Kirschfink et al. (1992)
Transplantation Proceedings
24: 2556-2557) reveals a need for a reliable and accurate assay system for monitoring in vivo C-activation in patient populations.
The complement system is made up of an array of enzymes and non-enzymatic proteins and receptors. The enzymes include a group of seven serine proteases: factor D, C1r, C1s, MASP, factor B, C2 and factor I. Complement activation occurs by one of three primary modes known as the “classical” pathway, the “alternative” pathway and the lectin pathway (see e.g., Ember et al. (1997)
Immunopharmacology
38:3-15).
These pathways are distinguished by the processes that initiate complement activation. The classical pathway is initiated by antibody-antigen complexes or aggregated forms of immunoglobulins; the alternative pathway is initiated by several ways, including spontaneous cleavage of a thioester, by certain structures on microbial and cell surfaces, such as amino groups, hydroxyl groups, and by water, and the lectin pathway, which is an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL, also designated mannan-binding protein) to carbohydrates (see, e.g., Thiel et al. (1997)
Nature
386:506-510).
Complement Pathways
Classical
The classical pathway is initiated by the binding of the first complement component (C1) to immune complexes through C1q, a subcomponent involved in binding to antibody. The c1 complex is composed of C1q and two homologous serine proteases, C1r and C1s (1:2:2 molar ratio). After binding to the immune complexes C1q undergoes a conformational change resulting in the conversions of C1r and C1s to their activated forms. Activated C1s cleaves C4 and C2 to generate a complex of their fragments C4b2a, which in turn cleaves C3 into C3a and C3b. C3b binds to immune complexes.
Alternative
The alternative pathway is activated by microbes without involvement of antibody. C3b molecules generated from C3 by interaction of C3 with two serine proteases, factors B and D, are deposited on the microbial surface where activation of C3 is amplifier. C3b produced by activation of either pathway acts as a central molecule in the subsequent formation of membrane attack complexes that can lyse microbes and also as an opsonin.
Lectin
Another of complement activation, called the lectin pathway (see, Reid (1998) in The human complement system in health and disease; Volanakis et al., Eds., pp. 33-48, Marcel Dekker, Inc., New York) exists. This pathway involves a mannan-binding protein (MBP), also designated mannose-binding lectin (MBL), that is identical to the bactericidal Ra-reactive factor that binds to the Ra polysaccharides on various strains of bacteria (Ji et al. (1993)
J. Immunol.
150:571-578). MBP is a multi-chain, multi-subunit protein that functions in a similar manner to the C1q component of the classical pathway. There are two proteinases associated with MBL called mannose-binding protein associate serine proteinases or MASP-1 and MASP-2 (see, e.g., Thiel et al. (1997)
Nature
386:506-510; see, also Takayama etal. (1994)
J. Immunol.
152:2308-2316). The MBL-MASP-1-MASP-2 complex is activated via MBL binding to neutral sugars resulting in activated MASP-2 enzyme which then cleaves component C4, and possibly the components C2 and C3, to initiate the classical complement pathway.
The collectin MBP, with its associated proteases, has the ability to activate complement and to act as an opsonin (a serum substance, that coats particulates such as viruses to promote phagocytosis). MBP-mediated complement activation is triggered by viruses and other pathogens and stimuli on which neutral sugar residues are exposed (see, Reid et al. (1998)
Immunology Today
19:56-59). In particular, MBP binds to carbohydrates on microbial and viral surfaces. This pathway differs from the classical and alternative pathways of complement activation. Complement activation via this pathway is mediated by an MBP complex. MBP is associated with serine proteases designated MBP-associated serine proteases (MASP-1 and MASP-2). The complex has C4- and C3-activating capacities upon binding to mannan. The complex contains two serine proteases MASP-1 and MASP-2 linked by a disulfide bond. In this form, MASP is capable of cleaving C4 and C3. The MBP-MASP-mediated complement cascade accompanied by C4 and C3 activation is distinct from the classical and alternative pathways and is designated the lectin pathway.
MBL is structurally related to the complement C1 subcomponent C1q and appears to activate the complement system through an associated serine proteases MASP-1 (see, e.g., Sato et al. (1993)
International Immunol.
6:66-669) and MASP-2 (see, e.g., Thiel et al. (1997)
Nature
386:506-510). MBL binds to specific carbohydrate structures on the surface of microorganisms, including bacteria, yeast, parasitic protozoans and viruses, and exhibits anti-bacterial activity through lytic complement components or by promoting phagocytosis.
Relationships among the pathways
These pathways are important components of host immune response to bacterial and viral infection. The classical pathway attenuates humoral response and is initiated by antibody antigen complexes. The alternative pathway is represents the first line of defense and is activated by a variety of macro

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