Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.
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PMID:Role of cathepsin D in antigen presentation of ovalbumin. 132 88

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.
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PMID:Different roles for thiol and aspartyl proteases in antigen presentation of ovalbumin. 169 78

We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.
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PMID:T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D. 171 25

MHC class II (MHC-II) molecules bind fragments of exogenous Ags in an intracellular endocytotic compartment. In view of divergent data on the MHC-II distribution in different cell lines, it was of interest to localize MHC-II molecules in a natural and the most potent APC type, the dendritic cell (DC). By using immunogold labeling of ultrathin cryosections of cultured mouse spleen DC, we found that MHC-II molecules were present abundantly at the plasma membrane and in intracellular compartments containing internal membrane vesicles and/or membrane sheets. The majority of these compartments was situated late in the endocytotic route, as demonstrated by the late appearance (after a lag of 30 min) of internalized exogenous tracer. These compartments contained the lysosomal enzymes cathepsin D and beta-hexosaminidase, but lacked the late endosomal marker cation-dependent mannose-6-phosphate receptor. We conclude that most of the intracellular MHC-II molecules in cultured spleen DC reside in a compartment with (pre)lysosomal characteristics, resembling the so-called MHC-II-enriched compartments (MIIC), originally described in B cells. We also investigated whether the presence of MHC-II molecules in endocytotic compartments was related to the kinetics of Ag processing and presentation by these cells. Pulse-chase endocytosis experiments with hen egg lysozyme (HEL) as a model Ag showed that activated spleen DC were able to efficiently process and present this Ag to an HEL-specific T hybridoma cell line. However, presentation started only after a lag of 2 h and was maximal after 6 h. The difference in time between the arrival of Ag in proteolytic endocytotic compartments, in particular MIIC, and effective Ag presentation is discussed in the context of DC maturation.
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PMID:MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells. 775 23

We compared the processing and presentation of the model Ag, hen-egg white lysozyme (HEL) expressed in C3.F6 APC as a fusion protein to three different acid hydrolases: cathepsin D, to an unglycosylated form of cathepsin D, and to pepsinogen. As expected from the biology of mannose 6-phosphate (Man-6-P)-containing enzyme, cathepsin D-HEL was delivered to the endosomal/lysosomal system. In contrast, the unglycosylated cathepsin D-HEL was retained in ER/Golgi and some was found in lysosomes. Most of pepsinogen-HEL was rapidly secreted from the APC. All transfectants presented HEL epitopes to T cell hybridomas. Regardless of the main route of traffic of the proteins, the strong I-Ak binding epitope HEL 48-62 was well presented by all. The biochemical forms of this epitope were identical for all. Three other epitopes of HEL that bind I-Ak with less affinity were processed equally well by unglycosylated cathepsin D-HEL and HEL-Ld. The glycosylated cathepsin D-HEL was less efficient in generating the 114-129 epitope. Pepsinogen-HEL was the less efficient of all transfectants in presenting these subdominant epitopes. Soluble cathepsin D-HEL recovered from culture supernatant was strongly immunogenic when added to C3.F6. The uptake was inhibited by free Man-6-P, indicating that the surface Man-6-P receptor can effectively deliver proteins to the class II MHC system.
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PMID:Presentation on class II MHC molecules of endogenous lysozyme targeted to the endocytic pathway. 905

Gallium arsenide (GaAs) is a semiconductor utilized in the electronics industry. Chemical exposure of animals causes a local inflammatory reaction, but systemic immunosuppression. Mice were administered i.p. 200 mg/kg GaAs crystals or latex beads, or vehicle. Five days after exposure, splenic macrophages were defective, whereas thioglycolate-elicited peritoneal macrophages (PEC) were more efficient in processing the Ag, pigeon cytochrome c, than vehicle control macrophages. Various aspects of the MHC class II Ag-processing pathway were examined. Both macrophage populations normally presented a peptide fragment to the CD4+ T cells. Surface MHC class II expression on the PEC was up-regulated, but splenic cells had normal MHC class II expression. PEC had elevated levels of glutathione and cysteine, major physiologic reducing thiols. However, the cysteine content of splenic macrophages was diminished. Proteolytic activities of aspartyl cathepsin D, and thiol cathepsins B and L were decreased significantly in splenic macrophages. On the other hand, thiol cathepsin activities were increased selectively in PEC. Latex bead-exposed PEC were not more potent APC, and their thiol cathepsin activities were unchanged, indicating that phagocytosis and nonspecific irritation were not responsible. The phenotype of PEC directly exposed to GaAs mirrored cytokine-activated macrophages, in contrast to splenic macrophages from a distant site. Therefore, GaAs exposure differentially modulated cathepsin activities in splenic macrophages and PEC, which correlated with their Ag-processing efficiency. Perhaps such distinct alterations may contribute to the local inflammation and systemic immunotoxicity caused by chemical exposure.
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PMID:Gallium arsenide modulates proteolytic cathepsin activities and antigen processing by macrophages. 972 6

We have previously prepared beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates with water-soluble carbodiimide and achieved reduced immunogenicity of beta-LG. In the present study, to elucidate the mechanism for the reduced immunogenicity of beta-LG, we investigated changes in the T cell response to beta-LG after conjugation with CMDs differing in molecular weight (about 40 and 162 kDa). Lymph node cells from BALB/c, C3H/He, and C57BL/6 mice that had been immunized with beta-LG or the conjugates were stimulated with beta-LG, and the in vivo T cell response was then evaluated by BrdU (5-bromo-2'-deoxyuridine) ELISA as the ex vivo proliferative response. T cells from the conjugate-immunized mice showed a lower proliferative response than those from the beta-LG-immunized mice. T cell epitope scanning, using synthesized peptides, showed that the T cell epitope profiles of the conjugates were similar to those of beta-LG, whereas the proliferative response to each epitope was reduced. These results indicate that the lower in vivo T cell response with the conjugates was not due to induction of conjugate-specific T cells, but due to a decrease in the number of beta-LG-specific T cells. After the lymph node cells from beta-LG-immunized mice had been stimulated with beta-LG or the conjugates, the efficiency of the antigen presentation of the conjugate to beta-LG-specific T cells was evaluated by BrdU ELISA as the in vitro proliferative response. The antigen presentation of beta-LG to the T cells was reduced by conjugation with CMD. In addition, conjugation with CMD enhanced the resistance of beta-LG to cathepsin B and cathepsin D, which suggest that conjugation with CMD inhibited the degradation of beta-LG by proteases in APC and led to suppression of the generation of antigenic peptides including T cell epitopes from beta-LG. It is therefore considered that the suppressive effect on the generation of T cell epitopes reduced the antigen presentation of the conjugates and that this reduction led to a decrease in the number of beta-LG-specific T cells in vivo. As a result, the decreased help to B cells by T cells would have reduced the antibody response to beta-LG. We conclude that suppression of the generation of T cell epitopes by conjugation with CMD is important to the mechanism for the reduced immunogenicity of beta-LG.
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PMID:Modulation of the T cell response to beta-lactoglobulin by conjugation with carboxymethyl dextran. 1252 6

32 healthy fertile women (control group) and 94 women with different-severity external endometriosis (EE) were examined. The activity of cathepsin D, plasminogen, AT-III, protein C, acid alpha1-antitrypsin as well as the parameters of antiprothrombin, thrombin and prothrombin time were investigated in serum of venous blood. In EE, the below parameters were found to be essentially increased: thrombin time--35.69 +/- 5.77 sec (controls--20.2 +/- 0.8 sec, p < 0.05), prothrombin time--30.43 +/- 7.21 sec (controls--21.6 +/- 0.8, p < 0.05), concentration of alpha2 macroglobulin--489.3 +/- 39.71 mg/dl (controls--240.2 +/- 12.2 mg/dl, p < 0.001), and AT-III activity--127.37 +/- 7.13% (controls--98.2 +/- 2.56, p < 0.001); at the same time, the parameters of plasmin were decreased--108.00 +/- 5.28% (controls--169 +/- 18%, p < 0.01).
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PMID:[Specific features of protease and antiprotease activity of blood in women with external endometriosis]. 1588 Nov 25

The identification of specific protein markers for colorectal cancer would provide the basis for early diagnosis and detection, as well as clues for understanding the molecular mechanisms governing cancer progression. In this report, we describe the proteomic analysis of the samples of colorectal cancer corresponding to seven patients. We have used the highly sensitive two-dimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) for the identification of proteins differentially expressed in tumoral and neighboring normal mucosa. We have detected differences in abundance of 52 proteins with statistical variance of the tumor versus normal spot volume ratio within the 95th confidence level (Student's t-test; p < 0.05). Forty-one out of 52 analyzed proteins were unambiguously identified by matrix-assisted laser desorption/ionization-time of flight MS coupled with database interrogation as being differentially expressed in colorectal cancer. An ontology analysis of these proteins revealed that they were mainly involved in regulation of transcription (synovial sarcoma X5 protein, metastasis-associated protein 1), cellular reorganization and cytoskeleton (cytokeratins, vimentin, beta actin), cell communication and signal transduction (annexins IV and V, relaxin, APC), and protein synthesis and folding (heat shock protein 60, calreticulin, cathepsin D, RSP4) among others. Preliminary studies demonstrated that the differentially expressed proteins found by 2-D DIGE could be confirmed and validated by immunoblotting and immunohistochemistry analyses in those few cases where antibodies were available. We believe that the incorporation of more samples and new datasets will permit the definition of a collection of proteins with a potential interest as biomarkers for colorectal cancer.
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PMID:Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis. 1592 90