Alberto Marquet

dd-Carboxypeptidase-Transpeptidase and Killing Site of β-Lactam Antibiotics in Streptomyces Strains R39, R61, and K11

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of β-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, β-lactamase activity was not involved in susceptibility to β-lactam antibiotics.

Cloning, molecular analysis and differential cell localisation of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata (CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CACK‐encoded polypeptide shows an additional sequence of four amino acids at the carboxy‐terminal end, which produces a different folding of the fragment with the presence of an α‐helix instead of the β‐sheet usual in all the other species studied. A similar result is elicited at the amino‐terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmania species used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

Cloning of the gp63 surface protease of Leishmania infantum: Differential post-translational modifications correlated with different infective forms

The Leishmania cell surface virulence factor gp63 is a protease family that plays an important role in the survival of the parasite protozoon into the host macrophages. We have cloned and characterised the gp63 gene from L. infantum. The sequence analysis of the gene indicates the existence of a high degree of conservation with the other old world species L. major and L. donovani. The similarity is lower with new world species with the exception of L. chagasi which shows a strikingly high percentage of identity (99-100%). In L. infantum the gp63 gene expresses two polypeptides of 58 and 60 kDa, respectively, which show a similar proteolytic activity. The 60 kDa polypeptide is expressed during the whole life cycle of the promastigote form of the parasite with a moderate increase at the stationary phase of growth while the 58 kDa product, although slightly present in the logarithmic phase, notable increases its expression during the highly infectious stationary phase. RNA analysis showed that the presence in L. chagasi of these two polypeptides correlates with two RNA molecules and with the degree of parasite infectivity, whereas in the case of L. infantum a single 3 kb messenger RNA is detected through the whole promastigote life cycle. Our data indicate that in L. infantum, the differences in gene expression of the gp63 protease family according to parasite phase of growth seem to be due to a differential pattern of glycosilation of the polypeptides which correlates with the different infective forms of the promastigote form of the parasite.

Synoviocytes type A bind exogenous antigens recognized by antibodies present in rheumatoid arthritis

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA anybodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel or RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65‐kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142–156 from bovine albumin and residues 65 78 from human pro‐collagen α1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.

Autoantibodies from Rheumatoid Arthritis Patients Recognize Antigens on the Synoviocyte Surface

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immuno‐precipitated from extracts containing mainly macrophage‐like synoviocytes (type A) but not from extracts of homogeneous fibroblast‐like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non‐rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögrens syndrome, psoriatic arthritis, and Crohns disease showed a weak cross‐reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.

Substructure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus

SummaryDimethyl suberimidate and dithiobis (succinimidyl propionate) have been used to explore the nearest neighbor relationship of the subunits (α, β, γ and δ by decreasing molecular weight) of F1-ATPase or BF1 factor of Micrococcus lysodeikticus. Cross-linking with the two diimido esters inhibited the ATPase activity but this inhibition never exceeded 50% of the initial value. The cross-linking pattern of this BF1 factor, as revealed by sodium dodecyl sulfate gel electrophoresis, shows a relative low proportion of high molecular weight aggregates which move slowly than the heaviest subunit (α). They are resolved as three components of molecular weights 200,000, 130,000 and 100,000 in 5% acrylamide gels, plus an additional component (mol. wt 80,000) identified in 10% acrylamide gels. The other aggregate bands represent cross-linking products of the smaller subunits (γ and δ) that may travel to the conventional position of the heavier subunits.The subunit composition of the aggregate bands has been determined through the reversion of dithiobis (succinimidyl propionate) cross-linking of the BF1 factor by dithiothreitol and analysis in second dimension by gel electrophoresis. The results indicate that γ subunit can cross-link with itself and with each of the other subunits except β. The α subunit is also able to cross-link with itself and with the other subunits although to a minor extent than γ, and that δ2 aggregates are present. These results represent a specific pattern of cross-linking for this BF1 factor as compared to other F1 coupling factors. It suggests a certain asymmetry in the spatial organization of the major subunits of M. lysodeikticus F1-ATPase where the γ subunit must play a central role. A subunit stoichiometry α3 β3 γ2 δ2 is proposed for whole F1-ATPase which leads to a molecular weight 440,000 consistent with the 430,000 value estimated by sedimentation equilibrium at low speed. A tentative structural model of M. lysodeikticus BF1 factor is derived from these data. The significance of the results in relation to the possible generalization of the molecular architecture of F1 factors is discussed.

Isolation, partial characterization of the cytoplasmic membrane fraction of Streptomyces albus G and dd-carboxypeptidase localization

Summary1.Treatment of Streptomyces albus G with lysozyme (mg dry weight mycelium/mg lysozyme = 2–3/1) leads to the isolation of membrane fractions whose yields amount to 12–20% of the total celullar protein depending upon the age and state of the bacterial culture.2.The isolated fractions are composed of 52.7 per cent protein and 41 per cent lipid with minor amounts of hexose (2.3–2.4%), hexosamines (1.6–2.1%), RNA (2%) and DNA (0.45%). They possess a chemical composition similar to that of other membrane systems.3.NADH oxidase activities are associated with the membrane fractions from cells of 18–20 h of age. These activities are not detected in the membrane fractions from older cells.4.All membrane fractions shape small vesicles. Differences in size and shape are, however, found between membrane preparations from cells of distinct ages.5.dd-carboxypeptidase is not selectively localized in a membrane fraction of a certain age. The subcellular distribution of this activity is similar to that of other lytic endopeptidases of S. albus G.6.The amounts of dd-carboxypeptidase associated with the various membrane fractions are variable and never very high. The specificity of the dd-carboxypeptidase activity associated with membrane fractions differs from that of the soluble enzyme.7.These results are discussed in relationship with the carboxypeptidase-transpeptidase hypothesis (Leyh-Bouille et al., 1970).

Differential protein kinase C phosphorylation sites in the L17 ribosomal protein from Leishmania infantum.

Leishmania infantum, the protozoan parasite responsible for leishmaniasis in Europe, is capable of undergoing developmental changes in vitro and provides an excellent model for the study of cell differentiation processes. We have cloned the gene encoding the L17 ribosomal protein. The LiL17 protein family belongs to the macrolide binding site, related to the peptidyl transferase center of the ribosome. Its comparison with other members of the protein family shows several structural differences that may reflect functional variations. The protein kinase C phosphorylation sites display an intermediate pattern involving differences in location and type of residue with respect to all the species considered. Gene-structural analysis suggests the existence of two different encoding genes. The expression of the genes seem to be different with the distinct growth phases of the parasite.

Aggregated Human Immunoglobulins Bind to Modified Proteins

Human immunoglobulins treated at 55°C in vitro are able to interact with maleylated bovine serum albumin (mBSA), but not with unmodified BSA. Gel filtration experiments demonstrated that the mBSA binding is associated with a high molecular weight complex of aggregated IgG. This aggregated IgG with binding capacity for mBSA could also be generated in vitro by treatment of human IgG at 37°C or 40°C and by incubation with human neutrophils. Furthermore, IgG aggregates with binding activity for mBSA could be detected in untreated synovial fluids from rheumatoid arthritis patients, indicating that these complexes occur in vivo. The phenomenon of binding to aggregated IgG was extended to other modified proteins such as maleylated human serum albumin (mHSA), acetyl low density lipoprotein (Ac‐LDL) and BSA reacted with oxidized linolenic acid. Soluble forms of these modified proteins were able to compete for the interaction between aggregated IgG and surface‐bound mBSA. We also found that aggregated IgG enhanced the Ac‐LDL‐dependent foam cell formation. These findings suggest a role for aggregated IgG in the metabolism of oxidized proteins.

Partial characterization of membrane-associated proteinases from Micrococcus lysodeikticus.

SummaryWe have identified cytoplasmic and membrane-associated proteinases from Micrococcus lysodeikticus (M. luteus) by the use of 125I-labeled casein and insulin as substrates. The membrane-associated activities were released by shock washing. Proteolytic activities showed pH optima at slightly alkaline values and we have concentrated on the activities at pH 8.0. The total units of both proteolytic activities were higher in the cytoplasmic than in any other fractions but the situation was different when the results were expressed in terms of specific activity. The activities against casein and insulin were differentiated by the action of inhibitors, divalent metal ions, Arrhenius plots and dependence on ionic strength. On these grounds, it is proposed that the membrane-associated enzyme acting on insulin is a single thiol proteinase while the proteolysis of casein reflects the action of, at least, two enzymes (thiol proteinase and serine proteinase). The distinction between the casein and insulin degrading activities was confirmed by crossed-inhibition experiments and by their behaviour on gel chromatography and concentration-dependent experiments.The aggregating properties have hampered the purification of the enzymes. The present results raise doubts about the significance of preventing membrane damage and degradation of membrane proteins by the addition of indiscriminated proteinase inhibitors during membrane isolation and manipulation.