Identification of the promastigote surface protease in seven species of Leishmania

Twelve different strains of Leishmania, including L. major, L. donovani, L. infantum, L. tropica, L. mexicana, L. amazonensis, L. braziliensis, and L. enriettii were examined for the presence of an ectoenzyme structurally and functionally related to the promastigote surface protease found in L. major LEM 513. All strains examined possess a protease that is labelled by surface iodination of living promastigotes. The electrophoretic migrations of the labelled proteases are similar in all species showing distinct ectoprotease activity. In addition, proteases that cross-react immunologically with the polypeptide moiety of the surface protease of L. major LEM 513 were found in 10 strains. These proteases were in all cases labelled by surface radioiodination. Two of the strains, L. amazonensis and L. braziliensis, do not show a strict correlation between protease activity, surface iodination, and immunological cross-reactivity with the promastigote surface protease of L. major LEM 513, although both strains possess distinct neutral proteases with electrophoretic behavior similar to that of the enzyme of L. major. The amount of proteolytic activity detected at the surface of living cells depends on the strain tested, and correlates qualitatively with the amount of promastigote surface protease detected on zymograms. We conclude that the proteolytic activity found at the surface of Leishmania promastigotes is a common feature of the species infective for humans and that the promastigote surface protease described in this article is structurally and functionally conserved in Old and New World Leishmania.     

Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase

The effects of a variety of inhibitors suggested that the promastigote surface protease (PSP) of Leishmania might be a zinc metalloprotease. To investigate this possibility, we conducted atomic emission and absorption spectroscopic analyses, which show that PSP contains 1 atom of zinc per 63-kDa monomer. Further studies showed that the enzyme can be biosynthetically labeled with 65ZnCl2. The comparison of the amino acid sequence of Leishmania major PSP with nine other zinc metalloproteinases revealed significant similarity in the area of their zinc-binding sites. These data show clearly that the promastigote surface protease of Leishmania is a zinc metalloproteinase. Secondary structure analysis by circular dichroism spectroscopy indicates that PSP contains over 40% beta-strand and less than 20% alpha-helical structure. The molecular masses of amphiphilic PSP (152 kDa) and of hydrophilic PSP (142 kDa), determined by quantitative electron scattering, suggest that the purified enzyme occurs in solution, and presumably at the cell surface, as a non-covalent homodimer.     

Inhibition of natural killing and antibody-dependent cell-mediated cytotoxicity by the plasma protease inhibitor alpha 2-macroglobulin (alpha 2M) and alpha 2M protease complexes

Over the past few years increasing attention has been given to the relationship between the immune response and proteases. The aim of our present study was to examine the dose-response effect of purified alpha 2-macroglobulin (alpha 2M) with varying degrees of protease (trypsin) saturation on natural killing (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC). The results demonstrated that alpha 2M with 50% trypsin saturation (fast alpha 2M) was more inhibitory in both assays than alpha 2M with no bound protease (slow alpha 2M).     

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.     

Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response.

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.     

Binding of native alpha 2-macroglobulin to human group G streptococci.

Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.     

Inhibition of the Aeromonas salmonicida extracellular protease by alpha 2-macroglobulin in the serum of rainbow trout

One of the major pathogenic factors of Aeromonas salmonicida, the bacterium causing furunculosis in fish, is considered to be a protease secreted in the extracellular products. This protease can be inhibited by normal trout and rabbit serum but the factor responsible has not hitherto been identified. The present results demonstrate that the A. salmonicida protease is inhibited by the alpha 2-macroglobulin of rainbow trout serum. This inhibitor accounts for about 9% of the total trypsin inhibiting capacity of trout serum, is alpha-migrating in electrophoresis, is moderately stable on storage at -20 degrees C, is destroyed by heating at 45 degrees C, has no requirement for cations and is inactivated by methylamine. The A. salmonicida protease is resistant to inhibition by 91.9% of the total trout serum trypsin inhibitors, without inactivating them, and to human alpha 1-antiproteinase. It is proposed that trout alpha 2M may have a defensive function against furunculosis but the resistance of the bacterial protease to inhibition by the majority of the serum protease inhibitors may represent pathogenic adaptation.     

Rheumatoid-like synovitis in rabbits by intra-articular administration of alpha 2-macroglobulin . trypsin complexes and alkylamine-treated alpha 2-macroglobulin

The present animal experiments revealed that repeated intra-articular administration of alpha 2-macroglobulin . trypsin complexes caused a rheumatoid-like synovitis in not preimmunized rabbits. In early stages of synovitis, acute to subacute pathomorphological alterations were observed. In later stages, the chronic infiltration was accomplished by early onset of fibroplasia in some experimental joints. Similar lesions were caused by administration of methylamine-treated alpha 2-macroglobulin, an inactive alpha 2-macroglobulin (alpha 2-M), very like that produced in the trapping of a proteinase. In contrast, administration of native plasma alpha 2-M was ineffective, whereas active trypsin produced moderate inflammation. It is felt that the present experimental model resembles immunopathological processes in human arthritides, where the occurrence of alpha 2-M . proteinase complexes was verified.     

Characterization of binding of human alpha 2-macroglobulin to group G streptococci.

An interaction was observed between human alpha 2-macroglobulin (alpha 2M) and streptococci belonging to group A, C, and G. Of 27 group C and 19 group G streptococcal cultures, 13 and 14, respectively, bound 125I-labeled alpha 2M. Some group A streptococci also interacted with alpha 2M. A number of other bacterial species tested did not react with alpha 2M. The binding of 125I-labeled alpha 2M to group G streptococci was time dependent, saturable, and could be inhibited by unlabeled alpha 2M. Inhibition experiments indicated that the streptococcal binding site for alpha 2M differed from the receptors for immunoglobulin G, fibrinogen, aggregated beta 2-microglobulin, albumin, and fibronectin. The alpha 2M binding activity was remarkably sensitive to trypsin and heat treatment indicating its protein nature. Kinetic analysis indicated a homogenous population of binding sites. The number of binding sites per bacterial cell was estimated to be approximately 20,000.     

Quantification of free alpha 2-macroglobulin and alpha 2-macroglobulin-protease complexes by a novel ELISA system based on streptococcal alpha 2-macroglobulin receptors.

An ELISA test system has been developed for the quantification of the two distinct forms of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M): (i) free alpha 2M (functionally active), which is the electrophoretically slow form (alpha 2 MS), and (ii) the alpha 2 M-proteinase complex (functionally inactive), which is the electrophoretically fast form of alpha 2 M (alpha 2 MF). Discrimination between the two types of alpha 2 M was achieved using extracts of the two independent streptococcal strains, M1 and Sc1, which express receptors for alpha 2 MS and alpha 2 MF, respectively, in combination with a monoclonal antibody specific for alpha 2 M. The assay system described is easy and reliable and permits quantitation of alpha 2 MS and alpha 2 MF in complex biological samples such as plasma and cutaneous suction blister fluid.     

Molecular interactions of surface protein peptides of Streptococcus gordonii with human salivary components

Oral streptococci play a large role in dental biofilm formation, and several types interact as early colonizers with the enamel salivary pellicle to form the primary biofilm, as well as to incorporate other bacteria on tooth surfaces. Interactions of surface molecules of individual streptococci with the salivary pellicle on the tooth surface have an influence on the etiological properties of an oral biofilm. To elucidate the molecular interactions of streptococci with salivary components, binding between surface protein (SspB and PAg) peptides of Streptococcus gordonii and Streptococcus sobrinus were investigated by utilizing BIAcore biosensor technology. The analogous peptide [change of T at position 400 to K in SspB(390-402), resulting in the SspB(390-T400K-402) peptide] from S. gordonii showed the greatest response for binding to salivary components and inhibited the binding of Streptococcus sanguis by more than 50% in a competitive inhibition assay in a comparison with other SspB and PAg peptides. This peptide also bound to the high-molecular-weight protein complex of salivary components and the agglutinin (gp340/DMBT1) peptide (scavenger receptor cysteine-rich domain peptide 2 [SRCRP 2]). In addition, the SspB(390-T400K-402) peptide was visualized by two surface positive charges in connection with the positively charged residues, in which lysine was a key residue for binding. Therefore, the region containing lysine may have binding activity in S. gordonii and S. sanguis, and the SRCRP 2 region may function as a receptor for the binding. These findings may provide useful information regarding the molecular mechanism of early biofilm formation by streptococci on tooth surfaces.     

Interactions between cytokines and alpha 2-macroglobulin.

Cytokines are often thought of as short range, short half-life molecules. This view may now be challenged by recent findings that indicate that alpha 2-macroglobulin and antibodies to cytokines may alter cytokine kinetics in vivo. alpha 2-Macroglobulin, one of the major proteins in serum, can bind a wide range of physiologically important molecules. As Keith James reviews here, the interaction between alpha 2-macroglobulin and a number of cytokines has recently come under scrutiny, revealing a new and potentially important mechanism for the modulation of cytokine activity, while in the following paper a modulatory role for anti-cytokine antibodies is considered.     

Immunomodulation by alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes: the effect on the human T lymphocyte response

Alpha 2-macroglobulin (alpha 2M), various alpha 2M-proteinase complexes and methylamine-treated alpha 2M were added to human lymphocyte cultures stimulated with the specific antigen purified protein derivative of tuberculin (PPD), pokeweed mitogen (PWM) and anti-CD3. alpha 2M-trypsin diminished all reactions in a dose-dependent way. In the PPD-induced stimulation of peripheral blood mononuclear cells, both change of configuration and remaining proteinase activity contributed to the suppressive activity. Separate exposure of adherent cells or the highly purified T lymphocytes to alpha 2M-trypsin complexes indicated that the effect was mediated through adherent cells. Addition of indomethacin did not modify the results. In the interleukin 2 (IL2)-dependent stimulation of purified T lymphocytes by anti-CD3 the effect of alpha 2M-proteinase complexes was probably due to the digestion of IL 2 through remaining proteinase activity. As alpha 2M-proteinase complexes are formed at sites of inflammation, the multiple immunomodulatory effects of alpha 2M-proteinase complexes might contribute to the dysregulation of the immune system in inflammatory diseases.     

Preliminary studies on the synthesis of alpha2-macroglobulin by human lymphocytes in vitro

Immunoprecipitation studies with antisera to alpha₂-macroglobulin (α₂M) were undertaken on the supernatants and extracts of human peripheral blood lymphocytes cultured in serum free medium containing [¹⁴C]glutamine. These studies revealed that an appreciable amount of the [¹⁴C]glutamine was recovered in the α₂M precipitate, suggesting that this protein was synthesized by lymphocytes.