Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-23
2002-09-03
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S190100, C424S238100
Reexamination Certificate
active
06444446
ABSTRACT:
BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates to the fields of protein chemistry and immunology; in particular to the isolation and characterization of new calcium binding proteolipids, the encoding DNA and to methods of using the novel proteins for detection of calcifying bacteria in various pathological conditions such as dental calculus and heart valve calcification.
1.2 Description of Related Art
Numerous studies have implicated oral bacteria in the etiology of transient bacteremia and endocarditis (Everett and Hirschmann, 1977). In particular, some studies have indicated that
C. matruchotii
may play a role in the occurrence of bacterial endocarditis and in the calcification of bicuspid heart valves (Cohen et al., 1992; lakovidis et al., 1992).
Corynebacterium matruchotii
is a microbial inhabitant of the oral cavity associated with dental calculus formation. As early as 1925
, C. matruchotii
(previously known as
Leptothrix buccalis
and
Bacterionema matruchotii
) was shown to be present in calcified deposits scraped from teeth (Bulleid, 1925). Subsequently it was demonstrated that these calcium phosphate containing deposits were due to bacteria in the dental calculus and that their production was regulated by various environmental factors (Ennever, 1960; Wasserman et al., 1958; Zander et al., 1960). At the light and electron microscopic level, mineralization in these bacteria has been found to occur either intracellularly, as in
Actinomyces israeli, Escherichia coli, Streptococcus sanguis, Streptococcus mutans, Streptococcus salivarius
, and some strains of
C. matruchotii
, or extracellularly, as in Veillonella and the diphtheroids (Ennever et al., 1974; Lie and Selvig, 1974; Rizzo et al., 1962; Streckfuss et al., 1974; Wasserman et al., 1958). The mineralized deposits produce electron diffraction patterns similar to that found in mammalian bone (Boyan-Salyers et al., 1978b; Ennever et al., 1971; Gonzales and Sognnaes, 1960). Also similarly to bone formation (Anderson, 1969), initial deposition of hydroxyapatite in calcifying bacteria has been associated with membranes (Ennever et al., 1968; Ennever et al., 1971; Vogel and Smith, 1976) or membrane components (Boyan and Boskey, 1984; Boyan-Salyers et al., 1978b; Boyan-Salyers and Boskey, 1980; Ennever et al., 1972; Ennever et al., 1976; Ennever et al., 1979).
Calcification of
C. matruchotii
has been examined using a number of in vitro models (Boyan-Salyers et al., 1978b; Ennever et al., 1971; Lie and Selvig, 1974; Vogel and Smith, 1976). Because mineralization will not occur without an adequate calcium supply,
C. matruchotii
can be studied under either calcification-permissive or calcification-nonpermissive conditions (Boyan et al., 1984; Boyan-Salyers and Boskey, 1980; Ennever et al., 1971), making it an excellent model for studying mineralization in general, and microbial calcification in particular. The initial steps in apatite formation involve Ca
2+
-binding to acidic phospholipids, particularly phosphoinositides and phosphatidylserine (Boyan-Salyers and Boskey, 1980; Vogel et al., 1978), followed by the addition of inorganic phosphate and Ca
2+
to form apatite [Ca
10
(PO
4
)
6
] clusters that are converted by hydration to hydroxyapatite (Vogel and Boyan-Salyers, 1976). It is believed that the acidic phospholipids in the membrane associate with specific proteolipids to form a complex which directs the initial phases of the process (Boyan et al., 1992; Boyan and Boskey, 1984; Boyan-Salyers et al., 1978a; Ennever et al., 1976; Raggio et al., 1986; Vogel and Boyan-Salyers, 1976).
Previous studies have demonstrated that calcifiable proteolipids isolated from
C. matruchotii
are involved in ion translocation across lipid bilayers. Using reconstituted bacteriorhodopsin-proteoliposomes, translocation of ions across the membrane was greatly enhanced in the presence of proteolipids extracted from
C. matruchotii
(Boyan et al., 1992; Swain et al., 1989; Swain and Boyan, 1988). Ion-transport across the liposomal membrane was inhibited by dicyclohexylcarbodiimide (DCCD, an inhibitor of proton channels). It has been suggested that proteolipids form an ionophore that could play a role in the intracellular accumulation of calcium and phosphate ions or export of protons, followed by initial mineralization on the inner leaflet of the membrane (Boyan et al., 1989a; 1989b; 1992; Swain and Boyan, 1988;1989).
A number of studies have shown that calcifiable bacteria contain constituents which can support calcification under appropriate conditions. Membranes isolated from
C. matruchotii
provide nucleating foci for apatite formation in vitro (Ennever et al., 1976; Vogel and Smith, 1976). More recent data indicate that specific calcifiable proteolipids a permit the ordered structuring of phospholipids in the cell membrane so that calcium-acidic phospholipid-phosphate complexes (CPLX) can form (Boyan et al., 1992; Boyan and Boskey, 1984; Boyan-Salyers and Boskey, 1980; Raggio et al., 1986).
Previous work has reported a 8-10 kDa proteolipid involved in
C. matruchotii
calcification (Boyan, 1985). In an earlier paper, phospholipids were reported to be associated with the protein moiety through hydrophobic interactions (Ennever et al., 1973); with partial removal of this phospholipid resulting in loss of calcifiability (Ennever et al., 1978a; 1978b). Later studies demonstrated the presence of additional proteolipids in the bacteria (Swain et al., 1989), which enhanced ion transport across liposomal membranes.
It has been suggested that proteolipids might function in two capacities during calcification: as sites for CPLX formation and in transport of Ca
2+
and P
i
to the calcification site or in the transport of protons away from the site (Boyan et al., 1989a; Swain and Boyan, 1989).
Proteolipids have been reported to play a role in both calcium binding in growth plate cartilage matrix vesicles (Cao et al., 1993; Genge et al., 1991;1992) and phosphate binding and transport over kidney brush border membranes (Debiec and Lorenc, 1988; Kessler et al., 1982;1988). In matrix vesicles a nucleational core complex, reminiscent of CPLX, has been reported, consisting of a membrane associated complex of Ca
2
+, P
i
, phosphatidylserine and annexins, proteins exhibiting proteolipid-like characteristics, and capable of initiating nucleation (Genge et al., 1991; Wu et al., 1993). On the other hand, phosphate transport across kidney brush border membranes has been associated with phosphorin, a 3 kDa membrane proteolipid (Kessler et al., 1982), as well as with a proteolipid-like Na+,Pi-binding protein with a molecular mass of 155 kDa (Debiec and Lorenc, 1988).
SUMMARY OF THE INVENTION
The present invention relates to the isolation and characterization of a novel proteolipid “bacteriocalcifin”, from
C. matruchotii
that is involved in the formation of dental calculus (“plaque”) and heart valve calcification. The new proteolipid represents a new class of calcium binding species designated “bacteriocalcifins”.
The present invention provides biologically active proteolipids comprising the amino acid sequences of bacteriocalcifin-1(SEQ ID NO:1) (5.5 kilodalton proteolipid, designated “bacteriocalcifin-1”) and bacteriocalcifin-2 (SEQ ID NO:2) (7.5 kilodalton proteolipid), as well as the nucleotide sequence of the bcf-1 gene for the 5.5 kilodalton proteolipid of (SEQ ID NO:3) and (SEQ ID NO:6) that includes the sequence of (SEQ ID NO:3) encoding a 5.5 kDa bacteriocalcifin. Among the biological properties of the bacteriocalcifins in the present invention is the capability to induce the formation of hydroxyapatite in vivo and the binding of calcium in an in vitro assay system.
An important aspect of the invention concerns the isolation, characterization, amino acid sequencing, cloning and nucleotide sequencing of the proteolipid from
C. matruchotii
, as well as the assay to determine in vitro calcification activity. The invention also contains the generation of polyclonal and m
Boyan Barbara D.
Dean David D.
Dijk Simon Van
Board of Regents , The University of Texas System
Duffy Patricia A.
Williams, Morgan and Amerson
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