Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH(2)-terminal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH(2)-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I(SC)). Peptides with as few as 16 amino acid residues are able to stimulate I(SC), although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I(SC) values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH(2)- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I(SC) are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I(SC). Palindromic peptide sequences based on both the NH(2)- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH(2)-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

Structure of high density lipoprotein. The immunologic reactivities of the COOH- and NH2-terminal regions of apolipoprotein A-I.

Only 5 to 10% of the apolipoprotein A-I (ApoA-I) of intact high density lipoprotein (HDL) is detectable by radioimmunoassay. In addition, when isolated ApoA-I is recombined with lipids in vitro, its immunologic reactivity is decreased by 30 to 95%. Thus, ApoA-I is less reactive immunologically in the presence of lipids. Our aim was to ascertain whether the COOH- or NH2-terminal regions of ApoA-I were equally reactive in intact HDL2. CNBr fragments of ApoA-I were produced by the method of Baker et al. (Baker, H.N., Jackson, R.L., and Gotto, A.M. (1973) Biochemistry 12, 3866-3871) and iodinated with lactoperoxidase. Double-antibody radioimmunoassays were set up using anti ApoA-I antisera and 125I-CNBr I (COOH-terminal region) or 125I-CNBr II (NH2-terminal). Both labels were bound by the antisera. Affinity columns were prepared by binding CNBr I or CNBr II to Sepharose 4B. Antibodies specific against CNBr I or CNBr II were isolated by means of these columns, suggesting that ApoA-I had at least two antigenic sites. In other assays using labeled fragments and anti ApoA-I antisera, 125I-CNBr I was displaced by CNBr I, ApoA-I , and HDL2 but not CNBr II. Conversely, 125I-CNBr II was displaced by CNBr II, ApoA-I, and HDL2 but not by CNBr I. Thus the assays were region-specific. The reactivities of isolated ApoA-I and the ApoA-I in intact HDL2-ApoA-I) were compared in these assays. On a molar basis, HDL2-ApoA-I was consistently more reactive (2- to 5-fold) in the 125I-CNBr I than in the 125I-CNBr II assays. The findings suggest (a) that the two terminal regions of ApoA-I are immunologically distinct, (b) that the two regions can be assayed independently of each other in intact HDL2, and (c) that the COOH-terminal region is more reactive immunologically than is the NH2-terminal. The results are compatible with a more "exposed" position for the COOH-terminal region on the surface of HDL2.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit,Requires Complex Formation of β4 and HD1/Plectin,and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH₂- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH₂-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.     

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.     

Dissociation between right atrial pressure and plasma atrial natriuretic factor following prolonged high salt intake

The influence of prolonged high salt intake on intravascular volume, right atrial pressure, plasma atrial natriuretic factor, and extra-atrial tissue (lung, kidney, and liver) COOH- and NH2-terminal atrial natriuretic factor content was investigated in normotensive rats. Despite prolonged high salt (8% NaCl) intake for 5 weeks, total intravascular volume was not impaired. However, right atrial pressure was increased by 54% (p less than 0.01) after salt loading. Although this increment in right atrial pressure should favor atrial natriuretic factor release after NaCl intake, plasma atrial natriuretic factor (COOH-terminal) concentrations markedly decreased from 97.8 +/- 27 to 38.9 +/- 8 pg/mL. Sodium and circulatory homeostasis was, however, well preserved. The lungs contained the highest levels of COOH- and NH2-terminal atrial natriuretic factor. Salt loading resulted in increased concentrations of low as well as high molecular weight atrial natriuretic factor in the lung but not in the kidney or the liver. Our study indicates a limited role of atrial natriuretic factor in adaptation to prolonged salt consumption in rats. Dissociation between right atrial pressure and plasma atrial natriuretic factor after salt intake implicates other factors regulating circulating peptide levels. Prolonged salt intake increases lung generation of atrial natriuretic factor.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.     

Phosphorylation of distinct regions of f1 histone. Relationship to the cell cycle.

The phosphorylation of different amino acids in distinct regions of f1 histone was studied in highly synchronized Chinese hamster cell populations (line CHO). The purified, 32P-labeled f1 histone was bisected into NH2-terminal and COOH-terminal fragments with N-bromosuccinimide. Tryptic phosphopeptides from these fragments were resolved using sequential high voltage electrophoretic steps on paper. No phosphorylation was observed in early G1-arrested cells. Interphase phosphorylation began in late G1 in the COOH-terminal portion of the molecule on serine. This event continued throughout S phase and persisted into mitosis. However, in mitosis additional phosphorylation was observed in the COOH-terminal portion of the molecule on threonine, and for the only time in the CHO cell cycle the NH2-terminal portion of the molecule was also phosphorylated on both serine and threonine. The peptide studies thus predicted that a minimum of four sites (two serine and two threonine) were phosphorylated in the f1 histone of mitotic CHO cells. This was confirmed using electrophoresis in long polyacrylamide gels.     

Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin.

Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.     

Identification and characterization of epitopes recognized by T lymphocytes and autoantibodies from patients with herpes gestationis

Autoantibodies associated with herpes gestationis (HG), a pregnancy-associated autoimmune skin disease, target the hemidesmosomal protein BP180. It was shown that the major noncollagenous stretch of the BP180 ectodomain (NC16A) harbors epitopes recognized by HG sera. Furthermore, Abs reactive with the homologous domain of murine BP180 are known to trigger a cutaneous blistering disease in mice by passive transfer experiments. The present study was aimed at characterizing the T cell responses and specificities of autoantibodies from two HG patients. Using immunoblotting and T cell proliferation assays, we have identified a 14-amino-acid stretch of the BP180 ectodomain (MCW-1; aa 507-520) that is recognized by both T cells and autoantibodies produced by the HG patients. The neonate born to one of these HG patients showed no signs of skin disease and had no detectable T cell response to the BP180 Ag, but did have a low titer of circulating anti-BP180 autoantibodies, presumably of maternal origin. BP180-specific T cell lines and clones developed from an HG patient specifically reacted with the MCW-1 epitope. The proliferative responses of these clones were restricted to HLA-DR, but not -DQ or -DP. These Ag-specific T cells expressed alpha/beta TCRs and a CD4 memory T cell phenotype and secreted IFN-gamma and IL-2, but not IL-4 or IL-6, suggesting that they are Th1-type lymphocytes. Further characterization of these Ag-specific T cells and autoantibodies will aid in elucidating the autoimmune mechanism(s) leading to the development of HG.     

Lipid-modified proteins as biomarkers for cardiovascular disease: a review.

Lipid-modified proteins are classified based on the identity of the attached lipid, a post- or co-translational modification required for their biological function. At least five different lipid modifications of cysteines, glycines and other residues on the COOH- and NH(2)-terminal domains have been described. Cysteine residues may be modified by the addition of a 16-carbon saturated fatty acyl group by a labile thioester bond (palmitoylation) or by prenylation processes that catalyze the formation of thioether bond with mevalonate derived isoprenoids, farnesol and geranylgeraniol. The NH(2)-terminal glycine residues may undergo a quite distinct process involving the formation of an amide bond with a 14-carbon saturated acyl group (myristoylation), while glycine residues in the COOH-terminal may be covalently attached with a cholesterol moiety by an ester bond. Finally, cell surface proteins can be anchored to the membrane through the addition of glycosylphosphatidylinositol moiety. Several lines of evidence suggest that lipid-modified proteins are directly involved in different steps of the development of lesions of atherosclerosis, from leukocyte recruitment to plaque rupture, and their expression or lipid modification are likely altered during atherogenesis. This review will briefly summarize the different enzymatic pathways of lipid modification and propose a series of lipid-modified proteins that can be used as biomarkers for cardiovascular disease.     

Endogenous lectin from cultured soybean cells: exposure of the SB-1 lectin on the cell wall

Digestion of seed soybean agglutinin with V-8 protease yielded seven distinct fragments (Mr 10,000-20,000) that were well-resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Each individual peptide (F1 through F7) was isolated; determination of the amino acid sequence at the NH2-terminal portion of each peptide established its position in the intact polypeptide of soybean agglutinin. The isolated peptides were used as affinity adsorbents to obtain antibodies that bound individual fragments (anti-F1 through anti-F7). These antibody preparations were, in turn, used in immunofluorescence staining of intact cultured soybean (SB-1) cells. Only those antibody preparations that bind to the NH2-terminal portion (residues 1-124) of the intact soybean agglutinin showed significant cell surface labeling. In contrast, the antibody preparations that bound to residues 125-253 failed to bind to intact SB-1 cells. These results suggest that the SB-1 lectin has the NH2-terminal portion of the polypeptide chain exposed and accessible at the cell surface, while the COOH-terminal portion of the same molecule may be masked, either through protein folding or through embedding in the cell wall. Limited digestion of the cell wall polysaccharides by cellulase or pectinase released the majority of the cell surface lectin.     

Identification and molecular cloning of two new 30-kDa insulin-like growth factor binding proteins isolated from adult human serum.

Three insulin-like growth factor binding proteins (IGFBP) with apparent molecular masses of 24, 28-30, and 30 kDa, nonreduced, have been isolated from human serum. The 15 NH2-terminal amino acids of the 24-kDa binding protein are identical with those of the 30-kDa BP. The apparent molecular mass of the latter is reduced to 24 kDa by N-glycanase, suggesting that the 30-kDa BP is the glycosylated form of the isolated 24-kDa BP. The complete amino acid sequences derived from the cloned cDNAs represent two new IGFBPs. They are tentatively termed IGFBP-4 and -5. The prepeptide sequences of BP-4 and -5 contain 27 and 21, the mature proteins 213 and 237 amino acids, respectively (Mr = 22,610 and 25,980). The NH2- and COOH-terminal thirds of BP-4 and -5 display pronounced homology to the other three human BPs. 16 of the 16-20 cysteines and 37 of the 213-289 amino acids (12.8-17.1%) are conserved in all five mature BPs. 10 amino acid positions located in the NH2-terminal region and shared by BP-1, -2, -3, and -5 are different in BP-4. These differences may account for the preferential affinity of BP-4 for IGF II. A most intriguing homology exists between the COOH-terminal quarter of the five IGFBPs, 10 repetitive domains of human thyroglobulin, a gastrointestinal tumor-associated antigen, and the invariant chain of the class II histocompatibility antigen. The cDNAs of five human IGFBPs are now available. They will allow their expression and production in sufficient quantities for in vivo studies to unravel their role in growth and metabolism.     

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.     

Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly

Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.     

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.     

Role of the Bullous Pemphigoid Antigen 180 (BP180) in the Assembly of Hemidesmosomes and Cell Adhesion—Reexpression of BP180 in Generalized Atrophic Benign Epidermolysis Bullosa Keratinocytes

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell–substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain α6β4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for α6 and β4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with α6β4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.