40-kD Fragment Research Articles

Abstract 91: Pathogenicity of Naturally Produced Fragments of Cardiac Myosin Binding Protein C and Their Effects on Heart Function and Failure

Background: Myosin binding protein C (MyBP-C) is a thick filament protein consisting of 1274 amino acid residues (149kD) and mutations in the cardiac isoform (cardiac MyBP-C; cMyBP-C) are responsible for a substantial proportion (20-35%) of identified cases of familial hypertrophic cardiomyopathy (FHC). Recently we found a 40kD fragment is produced from cMyBP-C when the heart is stressed, using a stimulus such as ischemia reperfusion injury. This fragment can be detected in both the mouse and human heart and appears to be stable. Its ability to interfere with normal cardiac function is unexplored. Methods and Results: To understand the potential pathogenicity of the 40kd fragment in vivo, we generated cardiac myocyte-specific transgenic mice (TG) using a Tet-Off inducible system to permit controlled expression in cardiomyocytes. When 40kD protein expression is induced by crossing the responder animals with tetracycline transactivator (tTA) mice, the double TG mice show protein expression and, subsequently, sarcomere dysgenesis and altered cardiac geometry. The double transgenic heart fails between 3 to 17 weeks of age. Expression, the fragment in cardiomyocytes led to development of significant cardiac hypertrophy with myofibrillar disarray and fibrosis. Subsequent analyses showed that MEK-ERK hypertrophic signaling pathways were activated. To determine the role of this pathway in the pathogenic response being generated, we subjected an experimental cohort of animals to treatment with the MAPK/ERK kinase inhibitor U0126 during pregnancy. The drug effectively improved heart function and prolonged survival as compared to the untreated control cohort. Conclusions: The data show that a 40kD fragment of cMyBP-C, which is generated during the development of heart disease in both the mouse and human, is a pathogenic fragment whose presence leads to hypertrophic cardiomyopathy and heart failure. Blockade of the MEK-ERK pathway was effective therapeutically in decreasing morbidity and increasing lifespan in the face of continued synthesis of the fragment.

Inhibition of breast cancer cell adhesion and bone metastasis by the extracellular adherence protein of Staphylococcus aureus

Bone metastasis is a common sequelae of breast cancer and the interaction of αvβ3-integrin with osteopontin (OPN) found in the extracellular matrix of mineralized tissues is implicated in this process. The integrin-dependent proadhesive and promigratory functions of OPN are particularly attributed to the 40kD N-terminal fragment that derives upon matrix metalloproteinase (MMP) cleavage. Based on the broad repertoire of interactions between Staphylococcus aureus extracellular adherence protein (Eap) and host components, we here characterized Eap to specifically interact with recombinant full-length OPN and the 40kD N-terminal MMP cleavage fragment, but not with the 32kD or the 25kD C-terminal fragments of OPN. Eap thereby prevented the OPN/αvβ3-integrin interaction, as well as the αvβ3-integrin-dependent adhesion of MDA-MB-231 breast cancer cells to full-length OPN or to the 40kD fragment and the migration of these cells towards OPN. Furthermore, Eap treatment markedly impaired the development of osseous metastasis of human MDA-MB-231 cells in vivo. Taken together, Eap may represent an attractive novel treatment for the prevention of breast cancer bone metastasis.

Detection of autoantibodies against thyroid peroxidase in serum samples of hypothyroid dogs

To establish a sensitive test for the detection of autoantibodies against thyroid peroxidase (TPO) in canine serum samples. 365 serum samples from dogs with hypothyroidism as determined on the basis of serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid-stimulating hormone, of which 195 (53%) had positive results for at least 1 of 3 thyroid autoantibodies (against thyroglobulin [Tg], T4, or T3) and serum samples from 28 healthy dogs (control samples). TPO was purified from canine thyroid glands by extraction with detergents, ultracentrifugation, and precipitation with ammonium sulfate. Screening for anti-TPO autoantibodies in canine sera was performed by use of an immunoblot assay. Thyroid extract containing TPO was separated electrophoretically, blotted, and probed with canine sera. Alkaline phosphatase-conjugated rabbit anti-dog IgG was used for detection of bound antibodies. TPO bands were observed at 110, 100, and 40 kd. Anti-TPO autoantibodies against the 40-kd fragment were detected in 33 (17%) sera of dogs with positive results for anti-Tg, anti-T4, or anti-T3 autoantibodies but not in sera of hypothyroid dogs without these autoantibodies or in sera of healthy dogs. The immunoblot assay was a sensitive and specific method for the detection of autoantibodies because it also provided information about the antigen. Anti-TPO autoantibodies were clearly detected in a fraction of hypothyroid dogs. The value of anti-TPO autoantibodies for use in early diagnosis of animals with thyroid gland diseases should be evaluated in additional studies.

Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.

Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells.

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.

Zinc is an essential cofactor for recognition of the DNA binding domain of poly(ADP-ribose) polymerase by antibodies in autoimmune rheumatic and bowel diseases.

To characterize autoantibody response to poly(ADP-ribose) polymerase (PARP) and to assess the significance of autoantibodies to the 2 zinc fingers of this enzyme in patients with autoimmune rheumatic and bowel diseases. The specificity of antienzyme autoantibodies was established by dot immunoassay with recombinant human PARP and by enzyme-linked immunosorbent assay using the recombinant N-terminal fragment containing the DNA binding domain of PARP, the recombinant C-terminal catalytic domain (40-kd fragment), a peptide containing the nuclear localization signal (NLS) of PARP, 2 synthetic peptides (and mutated peptides) corresponding to zinc-finger motifs F1 and F2 that are present in the DNA binding domain, zinc fingers from other self antigens (e.g., peptides from Ro60, Ro52, and U1C proteins), and poly(ADP-ribose). Sera from patients with autoimmune rheumatic and bowel diseases were tested, as were affinity-purified antibodies. Histocompatibility typing of systemic lupus erythematosus (SLE) patients was performed by serology. Antibodies from the patient sera reacted only weakly with the recombinant N- and C-terminal domains and with the NLS peptide. In contrast, the 2 synthetic peptides corresponding to zinc-finger motifs F1 and F2 represented immunodominant targets for IgG antibodies from patients with SLE, mixed connective tissue disease (MCTD), Crohn's disease, and ulcerative colitis. The sera from patients with SLE and MCTD showed much weaker reactivity with mutant peptides F1 and F2, which contain mutations at the cysteine residues involved in zinc coordination. F1/F2 antibodies did not cross-react with zinc fingers from other self proteins. No correlation was found between the presence of F1/F2 autoantibodies in SLE sera and the presence of other autoantibodies typical of this disease (e.g., anti-double-stranded DNA and poly[ADP-ribose] antibodies). The presence of F2 antibodies in the serum of SLE patients was negatively associated with HLA-DR6. An autoimmune response to PARP is potentially important because this enzyme is involved in DNA repair and is rapidly cleaved during the "execution phase" of apoptosis. The high prevalence in certain autoimmune rheumatic and bowel diseases of antibodies to F1 and F2, which are directly involved in this process, is further evidence implicating involvement of the DNA repair system in chronic inflammatory diseases.

Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis.

Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120-kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM-1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and prolonged activation of VLA-5 may mediate subsequent interactions with the basement membrane and extracellular matrix.

Binding sites for streptococci and staphylococci in fibronectin.

Purified cathepsin G fragments of fibronectin were used to locate the binding sites for streptococci and staphylococci in the fibronectin molecule. The iodinated, NH2-terminal, 30-kilodalton (kd) fragment bound to group A and G streptococci and to Staphylococcus aureus. The 125I-labeled, COOH-terminal, 120- to 140-kd fragment bound weakly to group A streptococcus strain and to S. aureus when tested in a buffer of low ionic strength. The 30- and 120- to 140-kd fragments inhibited the binding of iodinated fragments to bacteria. The two fragments were, on a molar basis, equally effective, and they were more potent inhibitors than intact fibronectin. The gelatin-binding 40-kd fragment neither bound to any of the bacterial strains nor inhibited the binding of 125I-labeled 30-kd or 125I-labeled 120- to 140-kd fragments to bacteria. The results indicate that fibronectin has at least two separate binding sites for streptococci and staphylococci, one in the NH2-terminal region and another in the COOH-terminal region of the molecule, both capable of specific interaction with a complementary structure exposed on streptococcal and staphylococcal cell surfaces.