Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-11-12
2001-08-21
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C530S326000, C530S395000, C536S023500
Reexamination Certificate
active
06277558
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns alpha-3 chain type IV bovine and human polynucleotides and peptides expressed by such polynucleotides which are useful in detecting Goodpasture antibodies and treating Goodpasture syndrome.
2. Background Information
The major structural component of mammalian basement membranes, type IV collagen, is composed of a number of distinct polypeptide chains (Timpl et al. 1981; Martin et at 1988; Timpl 1989). The most abundant species, &agr;1(IV) and &agr;2(IV) have been extensively characterized in man and mouse and an &agr; type chain from Drosophila also been identified (Soinmen et al. 1987; Blumberg et al. 1988; Hostikka and Tryggvason 1988; Savs et al. 1989; Muthukumaran et al. 1989). Characteristics of these collagens include a highly conserved carboxy-terminal noncollagenous (NC1) domain of ~229 residues, a shorter amino-terminal globular domain (7S domain) and a triple helical collagenous domain, in which interruptions occur in the Gly-Xaa-Xaa-Yaa repeat motif, giving a degree of flexibility to the triple helix. Within the membrane matrix the individual collagen chains exist as heterotrimer, which form a supra-molecular structure via interactions between the 7S domains of 4 molecules and the NCI domains of 2 heterotrimers (Timpl et al 1981).
Bacterial collagenase digestion releases the NCI domains from the other components of basement membrane as hexamers, comprised of the 3 NC1 domains from each of 2 interacting colllagen heterotrimers. The NCI domains can be further separated on the basis of molecular weight by denaturing polyacrylamide gel electrophoresis. This results in a number of separate monomeric and dimeric subunits (Mr=24,500-28,300 and 40,000-50,70000 respectively), including several which are distinct from the &agr;1(IV) and &agr;2(IV) chains (Butkowski et al. 1985; Wieslander et al. 1985). The monomeric subunits that result from collagenase digestion of human glomerular basement membrane (GBM) have been termed M24, M26, M28+++ and M28+, while the equivalent subunits of bovine basement membranes have been termed M1a, M1b, M2* and M3 (Kleppel et al. 1986; Butkowski et al. 1987). M24 (or M1a) and M26 (or M1b) are the NC1 domains of the &agr;1(IV) and &agr;2(IV) chains. M28+++ (or M2*) and M28+ (or M3) are the NCI domains of 2 novel collagen chains termed &agr;3(V) and &agr;4(V). Short segments of the junction between the collagenous and NCI domains of human and bovine &agr;3(IV) and &agr;4(IV) peptides have been sequenced, confirming that they have a type IV collagen structure (Saus et al 1988; Butkowski et al. 1990).
The &agr;3(IV) chain and the &agr;4(IV) chain are of particular interest as such chains have been implicated in the pathogenesis of Goodpasture syndrome and Alport-type familial nephtritis, clinical syndromes that affect GBM and cause functional kidney impairment (Hudson et al. 1989). Goodpasture syndrome is an autoimmune disorder characterized by glomerulonephritis, lung hemorrhage and anti-GBM antibody formation (Glassock et al. 1986). The nephritis and lung damage are mediated by these anti-GBM antibodies which are primarily targeted at the NC1 domain (M28+++) of &agr;3(IV) (Butkowski et at 1985; Wieslander et al. 1985; Kleppel al.1986). Alport syndrome is an inheritable disorder characterized by glomerulonephritis, sensorineural hearing loss and various abnormalities of the lens of the eye (Grunfeld, 1985). Ultrastructural GBM abnormalities frequently observed in the syndrome including thinning, diffuse splitting and multilamination of the lamina dense (Hinglais et al. 1972; Yoshikawa et al. 1981). Several investigators have reported that the GBM of some individuals with Alport syndrome does not react in vitro with Goodpasture antibodies nor with a monoclonal antibody that recognizes a Goodpasture epitope, suggesting that there is an abnormality of the &agr;3(IV) chain in these patients (Olsen et al 1980; Jervis et al. 1981; Jeraj et al. 1983; Kashtan et al. 1986; Savage et at 1986; Kleppel et al. 1987).
Recently a gene encoding another novel human type IV collagen chain, COL4A5, was cloned, on the basis of homology with the &agr;1(IV) and &agr;2(IV) chains (Hostikka et al. 1990; Myers et al. 1990. The existence of such a chain had not been expected from biochemical or immunological studies of GBM (glomerular basement molecular), and yet antibodies raised to a peptide fragment synthesized from the predicted amino acid sequence of &agr;5(IV) localized this chain to the GBM (Hostikka et at 1990). COL4A5 maps to Xq22, a region known from genetic linkage studies to contain a locus for Alport Syndrome (Atkin et al. 1988; Brunner et al. 1988; Flinter al. 1988). Further, COL4A5 has been shown to be musted in 3 of 18 large kindreds with the disease (Barker et al. 1990).
SUMMARY OF THE INVENTION
The present invention concerns an isolated and substantially pure polynucleotide enhancing 238 consecutive amino acids from the carboxy terminal end of the triple helical domain and all 233 amino acids of the carboxy terminal noncollageneous domain of the bovine &agr;3 chain of type IV collagen and a nucleotide sequence of said polynucleotide. The invention is also directed to a deduced amino acid sequence of the bovine &agr;3 chain of type IV collagen.
The present invention also relates to an isolated and substantially pure polynucleotide encoding 218 consecutive amino acids of the carboxy terminal noncollagenous domain of the human &agr;3 chain of type IV collagen and a nucleotide sequence of said polynucleotide. The invention is also directed to a deduced amino acid sequence of the human &agr;3 chain of the type IV collagen.
The above described polynucleotides can be used to express large amounts of proteins in vectors. Such proteins can be used to detect Goodpasture antibodies from the bloodstream of patients suffering from Goodpasture syndrome.
The present invention also concerns a peptide having no more than 218 amino acids of the human &agr;3 chain of type IV collagen comprising the following amino acid sequence:
ISRCQVCMKKRH (Iso Ser Arg Cys Gln Val Cys Met Lys Lys Arg His).
The invention also relates to 6 to all 12 consecutive amino acids of the sequence ISRCQVCMKKRH.
The invention also relates to a method for detecting Goodpasture antibodies from a bodily fluid or tissue from a patient, for example, a human, comprising contacting a bodily fluid or tissue from the patient, for example, a human, for example, contacting blood or a liquid fraction thereof, e.g. serum or plasma, with a peptide having no more than 218 amino acids of the human &agr;3 chain of type IV collagen comprising the following amino acid sequence: ISRCQVCMKKRH, whereby if Goodpasture antibodies are present a product will form of the antibodies and peptide and detecting for the presence of Goodpasture antibodies by, for example, by labelling the peptide, e.g., using an ELISA technique, i.e., using an enzyme label and detecting for the presence of the label on the antibody-peptide product.
The present invention is further directed to a therapeutic method of treating Goodpasture syndrome in a patient by neutralizing Goodpasture antibodies in the whole blood or liquid fraction thereof, e.g., plasma or serum, of the patient, for example, a human patient, by contacting the whole blood or liquid fraction thereof from the patient with an effective antibody neutralizing amount of a peptide having no more than 218 amino acids of the human &agr;3 chain of type IV collagen comprising the following amino acid sequence: ISRCQVCMKKRH. In such therapeutic method, the peptide is preferably bound to a solid support and the blood, serum or plasma from the patient passes over the peptide bound to the solid support, whereby the peptide captures the Goodpasture antibodies to remove such antibodies from the patient's blood, serum or plasma. The blood, serum or plasma with some, all or most of the Goodpasture antibodies removed is then returned to the bloodstream
Harper David S.
Kansas University Medical Center
Leary Louise N.
McDonnell & Boehnen Hulbert & Berghoff
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