Sally S. Twining

Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes

A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and subnanogram range using this assay. The assay is reproducible, has a low blank, and uses casein, which resembles natural substrates of most proteases.

The Serpin α1-Proteinase Inhibitor Is a Critical Substrate for Gelatinase B/MMP-9 In Vivo

We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.

A critical role for neutrophil elastase in experimental bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune skin disease characterized by subepidermal blisters and autoantibodies against 2 hemidesmosome-associated proteins, BP180 and BP230. The immunopathologic features of BP can be reproduced in mice by passive transfer of anti-BP180 antibodies. Lesion formation in this animal model depends upon complement activation and neutrophil recruitment. In the present study, we investigated the role of neutrophil elastase (NE) in antibody-induced blister formation in experimental BP. Abnormally high levels of caseinolytic activity, consistent with NE, were detected in extracts of lesional skin and blister fluid of mice injected with anti-BP180 IgG. The pathogenic anti-BP180 IgG failed to induce subepidermal blistering in NE-null (NE(-/-)) mutant mice. NE(-/-) mice reconstituted with neutrophils from wild-type mice became susceptible to experimental BP. Wild-type mice given NE inhibitors (alpha1-proteinase inhibitor and Me-O-Suc-Ala-Ala-Pro-Val-CH(2)Cl), but not mice given cathepsin G/chymase inhibitors (alpha1-antichymotrypsin or Z-Gly-Leu-Phe-CH(2)Cl), were resistant to the pathogenic activity of anti-BP180 antibodies. Incubation of murine skin with NE induced BP-like epidermal-dermal detachment. Finally, NE cleaved BP180 in vitro and in vivo. These results implicate NE directly in the dermal-epidermal cleavage induced by anti-BP180 antibodies in the experimental BP model.

Regulation of Proteolytic Activity in Tissues

Degradation of tissue proteins is controlled by multiple means. These include regulation of the synthesis of proteinases, activation of the zymogen forms, the activity of the mature proteinase, and the degradation of these enzymes and the substrates. Mature proteinases can be controlled by pH, calcium ions, ATP, lipids and the formation of complexes with other proteinases, proteoglycans, and inhibitors.

Alpha-1 proteinase inhibitor levels in keratoconus

The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.

Use of immunoadsorbents for the study of antibody binding to sperm whale myoglobin and its synthetic antigenic sites

Conditions for preparing immunoadsorbents of sperm-whale myoglobin and its five synthetic antigenic sites and for desorption of radiolabeled antibodies from the immunoadsorbents were studied. In immunoadsorbent titration studies, the sum of the amounts of antibodies bound in the plateau (maximum binding) by the adsorbents of the five sites accounted quantitatively for the entire (100%) antibody response to sperm-whale myoglobin.

Expression of wound healing and stress-related proteins in keratoconus corneas.

PURPOSE Keratoconus is characterized by thinning and scarring of the central portion of the cornea. This study was performed on keratoconus corneas to examine the expression of proteins related to wound healing including vimentin, an intermediate filament protein, and tenascin, and extracellular matrix protein. The expression of stress-related cytokines, heat shock proteins and ubiquitin was also investigated. METHODS Corneal buttons were collected from patients with keratoconus, normal subjects and patients with other corneal diseases such as pseudophakic bullous keratopathy. Immunofluorescence staining was performed on frozen sections for vimentin and tenascin, and immunoperoxidase staining was carried out on paraffin sections for cytokines, heat shock proteins and ubiquitin. RESULTS To varying degrees, all proteins examined, except tenascin and heat shock protein 90, were found to be expressed in normal human corneas. The expression of vimentin, tenascin, transforming growth factor-beta, interleukin-1, heat shock protein 27, and ubiquitin was enhanced in keratoconus corneas. A similar enhancement however was also observed in other diseased corneas. CONCLUSIONS Altered expression of several wound healing or stress-related proteins was noted in keratoconus corneas. The alterations appear to be nonspecific injury or wound responses in association with corneal diseases.

Genetic control of the immune response to myoglobin. IV. Mouse antibodies in outbred and congenic strains against sperm-whale myoglobin recognize the same antigenic sites that are recognized by antibodies raised in other species

The determination of the entire antigenic structure of sperm-whale myoglobin (Mb) was initially performed with antisera raised in rabbits and goats. Subsequently, we demonstrated that the synthetic antigenic sites were effective in stimulating in vitro mouse T-cell proliferation and that this proliferative response was under genetic control, with each antigenic site being controlled by a unique Ir gene. To determine unambiguously whether T-cells recognize the same molecular features as do B-cells, it was necessary to establish whether mouse antibodies are directed against these same sites. In the present work, using immunoadsorbent titration studies, these synthetic antigenic sites were shown to bind mouse 125I-labelled antibodies against Mb. With each of three outbred mice and four congenic strains, the total amounts of antibodies bound by the five sites accounted quantitatively for the entire antibody response against Mb. Moreover, with the four congenic strains, only the sites that were active in T-cell proliferation bound significant amounts of antibodies. It was concluded that (at least for Mb) the molecular features recognized by B-cells are also recognized by T-cells. These studies also confirm that the antigeniclty of the sites Is Independent of the immunized species and is inherent in their three-dimensional locations.

A pepsinogen from rainbow trout

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.

PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE

Currently, one-third of the worlds population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined. mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.