Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins.

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

Merlin differs from moesin in binding to F-actin and in its intra- and intermolecular interactions.

The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N- and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts with itself. Merlin 1 function in cells may thus depend on intra- and intermolecular interactions and their modulation, which include interactions with other members of this protein family.

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: identification by immunological and enzymological methods.

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.

Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method.

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.

Characterization of hemoglobin-hydrolyzing acidic proteinases in human and rat neutrophils.

The nature and levels of hemoglobin (Hb)-hydrolyzing acidic proteinases including cathepsin D and cathepsin E, which were most active at pH 3.5-4.0, were enzymatically and immunochemically compared between human and rat neutrophils. By subcellular fractionation and immunoprecipitation with discriminative antibodies specific for each enzyme, cathepsin D was shown to be present in the granular content fraction of both human and rat neutrophils and to account for about 35% of the total Hb-hydrolyzing activity. Cathepsin E was observed mainly in the cytoplasmic fraction of rat neutrophils from peripheral blood and peritoneal exudates and accounted for about 65% of the total activity, but it was not detected in human blood neutrophils. Immunoelectron microscopy on rat neutrophils revealed that cathepsin D was exclusively confined to lysosomes, whereas cathepsin E was localized mainly in the cytoplasmic matrix and often in the perinuclear spaces and the rough endoplasmic reticulum. The non-cathepsin D activity in human neutrophils, which represented about 65% of the total activity, appeared to be due to a serine proteinase, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride and was not inhibited by agents specific for aspartic-, cysteine-, or metallo proteinases. The enzyme(s) responsible for this activity was largely associated with the granular membranes, and a half of it could be described as an integral membrane protein on the basis of phase separation with Triton X-114 at 35 degrees C. The levels of these Hb-hydrolases in gingival crevicular fluid from human chronic inflammatory periodontitis patients were examined in order to clarify their participation in the periodontal tissue breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte medullasin levels in gingival crevicular fluid from chronic adult periodontitis patients and experimental gingivitis subjects.

The concentration of medullasin, an elastase-like serine proteinase, in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and experimental gingivitis subjects was determined by the highly sensitive immunoassay method. In periodontitis patients, the medullasin content increased with increase of the GCF volume and then attained a maximum value at a relatively mildly inflamed stage. The value was maintained through more serious stages of disease activity. However, the medullasin content was independent of the probing depth. The medullasin content of the patients was markedly decreased after periodontal treatment, indicating that the enzyme participates in the development of the chronic periodontitis. Large amounts of medullasin were also detected in GCF from experimental gingivitis subjects, although it was not detected by the activity measurements. There was a rapid increase in the medullasin content during the 4-day period after abstention from oral hygiene measures, which corresponded to those of severely inflamed periodontitis patients. The peak value decreased up to the 7th-d followed by a gradual increase during the 21-d experimental period. The increased medullasin level rapidly decreased following resumption of oral hygiene measures. The results suggest that medullasin plays important roles both in the defence mechanism against the gingival inflammation and in the development of the acute inflammation.

Cathepsins B, H and L activities in gingival crevicular fluid from chronic adult periodontitis patients and experimental gingivitis subjects.

To clarify roles of lysosomal cysteine proteinases cathepsins B, H and L in pathological destructive process of periodontal tissues, levels of their enzymatic activities were determined in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and from experimental gingivitis subjects. In periodontitis patients, higher levels of cathepsins B, H and L activities were found at sites with more serious signs of the disease activity. The total activity of each enzyme (per unit time) was positively correlated with the GCF volume. However, it had little or no correlation with the probing depth (PD). In contrast, the specific activity of each enzyme in GCF (activity units per mg protein), which reflects the selectivity of enzyme exudation, was negatively correlated with the GCF volume. These results suggest that the cysteine proteinases are selectively released into gingival crevices at a relatively mild stage of periodontitis. In experimental gingivitis subjects, no significant activity of each enzyme was detected in GCF, even when the quantity of GCF was comparable to that from periodontitis patients. These data suggest that no significant amounts of these enzymes are released at experimental gingivitis sites or that a homeostatic mechanism, including regulation by protease inhibitors, may control activities of these enzymes in GCF with acute inflammation.