Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: evidence for widespread distribution of the Tn antigen (GalNAc-Ser/Thr) and identification of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.     

Structural changes of low density lipoproteins with Cu2+ and glucose induced oxidation

The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors.     

Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.     

Immunization with cathepsin L proteinases CL1 and CL2 secreted by Fasciola hepatica elicit a preferential type 1 response based on IgG2a antibodies in rats

Cathepsin L proteinases (CL1 and CL2), the major components of Fasciola hepatica excretion/secretion products (E/S) are considered potential antigens of a vaccine against fascioliasis. The humoral response elicited by CL1 and CL2 in rats either immunized with the enzymes or infected with F. hepatica has been analysed, examining specific IgE and IgG subclass dynamics. The experiment was continued for 10 weeks and peripheral blood eosinophilia was also determined. Infected rats presented peaks of eosinophilia at weeks 3 and 7 post-infection, while those immunized with CL1 and CL2 were no different from controls. Total IgE in infected rats increased up to week 5, reaching 30 microg(-1) in some cases, then decreased slowly and rising again towards the end of the experiment. Determination of specific IgE, carried out in sera previously absorbed with Protein G-Sepharose, reached a peak in infected rats between weeks 2 and 5, depending on the individual. In immunized rats both total and specific IgE levels remained around the pre-immunization values. With regard to the IgG subclass responses to E/S products, in infected rats IgG1 predominated over IgG2a, and the reverse was true in rats immunized with CL1 and CL2 and tested against the respective antigens. In all cases an increase in IgG1 and IgG2a antibody titres was seen, with maximum levels being reached later (weeks 6-7) in infected rats than in immunized ones (weeks 4-5). No IgG2b or IgG2c responses were detected in any of the groups studied.     

Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Affinity for inorganic carbon of Gracilaria tenuistipitata cultured at low and high irradiance

Regulation by irradiance level of the mechanism for dissolved inorganic carbon (DIC) acquisition was examined in the red macroalga Gracilaria tenuistipitata Zhang et Xia. For this purpose, affinity for external DIC, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli grown at 45 and 500 μmol photons m⁻² s⁻¹. Oxygen evolution rates declined by 50% when the medium pH was changed from 8.1 to 8.7, and the pH compensation point attained was ca. 9.2. These characteristics were unaffected by the light treatments. In contrast, photosynthetic conductance for DIC at pH 8.7 was doubled in thalli grown at high irradiance compared with those grown at low irradiance (to 0.74 × 10⁻⁶ from 0.33 × 10⁻⁶ m s⁻¹). Photosynthetic rates at saturating DIC concentration were also higher by 60% in thalli grown at high irradiance. These differences could not be attributed to changes in the use of external DIC, since external CA activity did not vary. Although the irradiance level did not modify the pool size of Rubisco, Rubisco content expressed on a chlorophyll a basis was almost doubled at high irradiance. These results likely indicate that the internal transport of DIC towards the active-site of Rubisco, rather than the external use of DIC, is enhanced in the thalli grown at high irradiance. Received: 7 June 1999 / Accepted: 16 October 1999