Genetic relationships between efficiency traits and gut microbiota traits in growing pigs being fed with a conventional or a high-fiber diet.

In pigs, the gut microbiota composition plays a major role in the process of digestion, but is influenced by many external factors, especially diet. To be used in breeding applications, genotype by diet interactions on microbiota composition have to be quantified, as well as their impact on genetic covariances with feed efficiency (FE) and digestive efficiency (DE) traits. This study aimed at determining the impact of an alternative diet on variance components of microbiota traits (genera and alpha diversity indices) and estimating genetic correlations between microbiota and efficiency traits for pigs fed a conventional (CO) or a high-fiber (HF) diet. Fecal microbes of 812 full-siblings fed a CO diet and 752 pigs fed the HF diet were characterized at 16 weeks of age by sequencing the V3-V4 region of the 16S rRNA gene. A total of 231 genera were identified. Digestibility coefficients of nitrogen, organic matter, and energy were predicted analyzing the same fecal samples with near infrared spectrometry. Daily feed intake, feed conversion ratio, residual feed intake and average daily gain (ADG) were also recorded. The 71 genera present in more than 20% of individuals were retained for genetic analyses. Heritability (h²) of microbiota traits were similar between diets (from null to 0.38 ±â€…0.12 in the CO diet and to 0.39 ±â€…0.12 in the HF diet). Only three out of the 24 genera and two alpha diversity indices with significant h² in both diets had genetic correlations across diets significantly different from 0.99 (P < 0.05), indicating limited genetic by diet interactions for these traits. When both diets were analyzed jointly, 59 genera had h² significantly different from zero. Based on the genetic correlations between these genera and ADG, FE, and DE traits, three groups of genera could be identified. A group of 29 genera had abundances favorably correlated with DE and FE traits, 14 genera were unfavorably correlated with DE traits, and the last group of 16 genera had abundances with correlations close to zero with production traits. However, genera abundances favorably correlated with DE and FE traits were unfavorably correlated with ADG, and vice versa. Alpha diversity indices had correlation patterns similar to the first group. In the end, genetic by diet interactions on gut microbiota composition of growing pigs were limited in this study. Based on this study, microbiota-based traits could be used as proxies to improve FE and DE in growing pigs.

The link between the composition of the gut microbiota, i.e the composition of microorganisms in the gut, in pigs and their feed efficiency, i.e. their ability to utilize nutrients, as well as their ability to digest were studied from a genetic point of view. A family structure of 1,564 pigs were studied and fed with two different diets. One of the full-sib was fed a conventional diet used in breeding farms and the other one an alternative diet containing raw materials, less expensive but with a higher content of dietary fibers more difficult to digest. This study has shown that some microbiota microorganisms were genetically correlated with feed and digestive efficiency performances, positively or negatively, depending on the microorganisms. In addition, the diversity of microorganisms in the animal's gut was favorably correlated with the feed and digestive performances studied. Therefore, there is a genetic link between these performances and the composition of the animal's gut microbiota. Thus, a potential genetic selection on some intestinal microorganisms or diversity of microorganisms would allow to improve these performances, and in particular when pigs are fed with diet more difficult to digest.

Comparative study between fasciocutaneous and myocutaneous flaps in the surgical treatment of pressure ulcers of the sacral region.

INTRODUCTION: Decubitus ulcers of the sacral region are common conditions in bedridden patients. Deep lesions (Stages III and IV) often require surgical treatment for closure. Flaps of the region are the first choice for treatment. We present our experience in the treatment of these lesions and compare two different approaches: local fasciocutaneous flap and gluteus maximus myocutaneous flap with V-Y advancement. METHOD: From March 2009 to May 2014, 32 patients underwent closure of sacral pressure ulcers by flaps, 17 of them with rotational local fasciocutaneous flaps and 15 with myocutaneous flaps of the gluteus maximus muscle with V-Y advancement. Evolution regarding complications and rate of success after two months was compared between the groups. RESULTS: Out of the 32 operated patients we obtained resolution of lesions after two months in 23 (71.8%), 10 patients in the fasciocutaneous flap group (58.8%) and 13 cases in the myocutaneous flap group (86.6%). The most common complication was partial dehiscence of sutures in 12 patients (37.5%), 8 patients in the fasciocutaneous flap group (47%) and 4 patients in the myocutaneous flap group (26.6%). The group of patients reconstructed with local fasciocutaneous flaps presented 3 cases with seroma, one with hematoma and 6 with partial cutaneous necrosis; these patients also required more drainage time. CONCLUSIONS: Both the local rotational fasciocutaneous flap and the myocutaneous flap of the gluteus maximus muscle in V-Y flap can be used in the surgical treatment of sacral ulcers. In our experience, a reduced success rate and more complications were found in the local fasciocutaneous reconstructive method.

A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.

Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei.

African trypanosomes are coated by 10 million copies of a single variant specific glycoprotein (VSG) which are anchored in the plasma membrane by glycosylphosphatidylinositol (GPI). A GPI-specific phospholipase C (GPI-PLC) triggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular. One enigmatic aspect of GPI-PLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane. The data presented herewith disclose an unexpected possible solution for this puzzle: upon cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus becomes double coated. This unusual VSG rearrangement was stable in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but transiently preceded VSG release in wild-type parasites. The formation of double coat membrane (DCM) was independent of the presence or activation of GPI-PLC, occurred both at 4 degrees C and 30 degrees C and was unaffected by the classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (PCM). DCMs conserved the same coat thickness and association with subpellicular microtubules as in intact cells and were prone to form vesicles following gradual detachment of the latter. Our data also demonstrate that: (i) GPI-PLC expressed by one trypanosome only targets its own plasma membrane, being unable to release VSG of another parasite; (ii) DCMs concomitantly formed from trypanosomes expressing different VSGs do not intermix, an indication that DCM might be refractory to membrane fusion.

Conservation of genetic linkage between heat shock protein 100 and glycosylphosphatidylinositol-specific phospholipase C in Trypanosoma brucei and Trypanosoma cruzi.

The experiments described in this paper were designed to try and isolate a recombinant DNA clone encoding a Trypanosoma cruzi homologue of the Trypanosoma brucei glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) gene. Despite the ready biochemical detection of phospholipase C activities that hydrolyse GPI-anchors of cell surface proteins in T. cruzi, it did not prove possible to isolate any recombinant DNA clones using the T. brucei gpi-plc gene as a probe. On determining the DNA sequence to the 5' side of the gpi-plc gene it was found to be adjacent to a gene that encodes a 100 kDa heat shock protein (HSP100). To investigate whether this linkage between the hspl00 and gpi-plc genes was conserved in T. cruzi, a probe derived from the T. brucei hsp100 gene was used to isolate T. cruzi genomic clones. These were partially sequenced and shown to contain an hsp100 gene. Restriction enzyme fragments located to the 3' side of the T. cruzi hsp100 gene were then sequenced and found to contain a gene that encodes a polypeptide (TcPLC1) that has 46% amino acid sequence identity with the T. brucei GPI-PLC including most of the key residues involved in inositol binding and the catalytic histidine. A recombinant form of TcPLC1 was produced and shown to possess phospholipase C activity towards a GPI-substrate. Thus, the hsp100 and gpi-plc genes are adjacent in T. brucei and this linkage is conserved in T. cruzi. This observation has been used to facilitate the isolation of a clone encoding a T. cruzi phospholipase C gene.

An assay for glycosylphosphatidylinositol-anchor degrading phospholipases.

This paper describes a new approach to assay phospholipases which cleave glycosylphosphatidylinositol using a biotinylated protein substrate coupled to 125I-streptavidin and Triton X-114 phase separation. Substrate preparation with variant surface glycoprotein of Trypamosoma brucei, its characterization and solubilization by glycosylphosphatidylinositol-specific phospholipase C and D are reported. Hydrolysis of substrate exhibited first-order kinetics with respect to enzyme concentration, and the rate constant of the reaction is independent both from substrate concentration and reaction time. This assay was compared with the one using 3H-myristoylated variant surface glycoprotein and proved to be equally suitable to quantitate glycosylphosphatidylinositol-specific phospholipases, with the advantage that avoids biosynthetic labeling. Furthermore, it introduces a basic methodology which can be easily adapted to use other glycosylphosphatidylinositol-anchored proteins as substrates.

Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response.

A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.

Identification of complete precursors for the glycosylphosphatidylinositol protein anchors of Trypanosoma cruzi.

The survival of Trypanosoma cruzi, the causative agent of Chagas' disease, depends vitally on proteins and glycoconjugates that mediate the parasite/host interaction. Since most of these molecules are attached to the membrane by glycosylphosphatidylinositol (GPI), alternative means of chemotherapeutic intervention might emerge from GPI biosynthesis studies. The structure of the major 1G7 antigen GPI has been fully characterized by us (Güther, M. L. S., Cardoso de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J.(1992) J. Biol. Chem. 267, 6820-6828; Heise, N., Cardoso de Almeida, M. L., and Ferguson, M. A. J.(1995) Mol. Biochem. Parasitol. 70, 71-84), and based on its properties we now report the complete precursor glycolipids predicted to be transferred to the nascent protein. Migrating closely to Trypanosoma brucei glycolipid A on TLC, such species, named glycolipids A-like 1 and A-like 2, were labeled with tritiated palmitic acid, myo-inositol, glucosamine, and mannose, but surprisingly only the less polar glycolipid A-like 1 incorporated ethanolamine. The predicted products following nitrous acid deamination and digestion with phospholipases A2, C, and D confirmed their GPI nature. Evidence that they may represent the anchor transferred to the 1G7 antigen came from the following analyses: (i) alpha-mannosidase treatments indicated that only one mannose was amenable to removal; (ii) their lipid moiety was identified as sn-1-alkyl-2-acylglycerol due to their sensitivity to phospholipase A2 (PLA2), mild base and by direct high performance TLC analysis of the corresponding benzoylated diradylglycerol components; and (iii) both glycolipids incorporated 3H-fatty acid only in the sn-2- and not in the sn-1-alkyl position as previously found in the GPI of the mature 1G7 antigen. Based on the differential [3H]ethanolamine incorporation pattern and the recent report that an aminoethylphosphonic acid (AEP) replaces ethanolamine phosphate (EtNH2-PO4) in the GPI in epimastigote sialoglycoproteins (Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendonça-Previato, L.(1995) J. Biol. Chem. 270, 7241-7250) it is proposed that glycolipid A-like 2 contains AEP and A-like 1 EtNH2-PO4. In the in vitro cell-free system both glycolipids were synthesized simultaneously and do not seem to bear a precursor/product relationship. Among the various components synthesized in vitro a glycolipid C-like corresponding to a form of glycolipid A-like 1 acylated on the inositol was also characterized. Phenylmethylsulfonyl fluoride, an inhibitor known to block the addition of ethanolamine phosphate in T. brucei but not in mammalian cells, also inhibits the synthesis of glycolipids A-like and C-like in T. cruzi, indicating that the putative trypanosome EtNH2-PO4/AEP transferase(s) might represent a potential target for chemotherapy.

Paracoccidioides brasiliensis expresses both glycosylphosphatidylinositol-anchored proteins and a potent phospholipase C.

This study reports, for the first time, the detection of glycosylphosphatidylinositol (GPI) membrane anchors in proteins of a pathogenic fungus, Paracoccidioides brasiliensis. Taking into account that fungal antigens are found in the sera of paracoccidioidiomycosis patients and that cleavage of this glycolipid by phospholipases is a means of selective protein release, the presence of an enzyme with this property has also been investigated. Using a methodological approach in which the proteins were immobilized on nitrocellulose, treated with phospholipase C of Trypanosoma brucei and then probed with antibodies which recognize the 1,2-cyclic-phosphate inositol moiety formed as a reaction product in proteins bearing the glycolipid anchor, it was possible to detect a major glycoprotein in the 80- to 90-kDa range, as well as two other minor species of 66 and 43 kDa. All of them bind to Concanavalin-A and are also substrates of a very potent fungal phospholipase C which is inhibited by p-chloromercuri-phenylsulfonic acid and is insensitive to EDTA. The integrity of glycosylphosphatidylinositol anchors in proteins of P. brasiliensis is impaired by 0.1 M NaOH, a finding indicative of a diacyl glycerolipid moiety which is quite surprising since it is, with the exception of African trypanosomes surface proteins and Torpedo acetylcholinesterase, an uncommon feature among GPIs in general. The present findings may have implications in the pathology of paracoccidiodomycosis.

Characterization of the lipid moiety of the glycosylphosphatidylinositol anchor of Trypanosoma cruzi 1G7-antigen.

The 90-kDa stage-specific 1G7-antigen has been implicated in the invasion of host cells by the metacyclic forms of Trypanosoma cruzi. The antigen is attached to the plasma membrane via glycosylphosphatidylinositol, the partial structure of which was the first to be determined for a protein of this parasite. In this study, the complete structure of the lipid component of the anchor was determined by electrospray mass spectrometry, gas chromatography mass spectrometry, phospholipase sensitivity and high-performance thin-layer chromatography of the diaradylglycerol components after benzoylation. These analyses showed that the lipid moiety of 1G7-antigen is composed essentially of 1-O-hexadecyl-2-O-hexadecanoyl-phosphatidylinositol and 1-O-hexadecyl-2-O-octadecanoyl-phosphatidylinositol. The high sensitivity of the electrospray mass spectrometric analysis unexpectedly revealed the presence of a small proportion of putative inositol-phosphoceramide structures, and confirmed the absence of inositol-acylated species. An interesting finding was that the biosynthetic incorporation of [3H]palmitate labelled solely the acyl position, and not the 1-O-alkyl chain in the 1G7-antigen anchor.

The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting.

Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.

Expression of Mycobacterium leprae 18-kDa antigen in yeast in a GPI-anchored form.

The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.

Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi.

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phospatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuringiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera.

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4.

Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4

Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi.

We have previously shown that 35- and 50-kDa glycoconjugates of cultured metacyclic trypomastigotes participate in the attachment of parasites to mammalian cells. Here we show that when metacyclic trypomastigotes are incubated with [3H]sialyllactose, most of the sialic acid is transferred to these 35/50-kDa molecules in a reaction catalyzed by a parasite transsialidase. The sialic acid is incorporated in oligosaccharides of about 10 glucose units in size that are released from the glycoconjugate by mild alkaline hydrolysis. Compositional analysis reveals that the 35/50-kDa molecules are highly glycosylated proteins rich in threonine, galactose, N-acetyl-glucosamine and sialic acid. These glycoproteins can be labeled in vivo with [3H]palmitate, and the labeled fatty acid is released by glycosylphosphatidylinositol specific phospholipases C. This result, associated with the fact that they contain mannose, ethanolamine, myo-inositol, and lipid, indicate that these glycoproteins are anchored to the membrane by glycosylphosphatidylinositol. During cell invasion, these molecules appear to be capped and locally released by the parasite.

Proteins anchored via glycosylphosphatidylinositol and solubilizing phospholipases in Trypanosoma cruzi.

The presence of GPI anchors and phospholipases capable of solubilizing them in Trypanosoma cruzi has been investigated in epimastigotes, metacyclic trypomastigotes from axenic cultures and tissue culture trypomastigotes. The GPI anchored proteins in epimastigote forms are scarce when compared to their abundance in the parasite forms which can infect mammals, and GPI-solubilizing phospholipases C have been found in all life cycles stages. In epimastigote and metacyclic forms, the activity is found in the soluble fraction upon cell lysis, whereas in tissue cultured trypomastigotes it is membrane bound and, being mostly sensitive to p-chloromercuriphenylsulfonate, resembles closely the GPI specific phospholipase of Trypanosoma brucei. Sequential immunoprecipitations with monoclonal antibodies and anti-CRD indicated the presence of several sub-populations among the surface proteins of metacyclic trypomastigotes, five of these belonging to the GPI-anchored 90 kD family. Among this family, the epitopes recognized by MAb-1G7 are present in three members, one of them also expressing the 3F6 epitope. There are 2 members recognized only by MAb-3F6 but not by MAb-1G7, one of them being probably galactosylated on the GPI since it can be immunoprecipitated by anti-CRD. Very strangely, the epitope recognized by the MAb-WIC29.26 was always present on the gp72, as originally described, but under certain circumstances appeared cryptic on one of the 90 kD species. During epimastigote transformation into metacyclic trypomastigotes in vitro, the ability of the GPI of the 1G7-antigen to be solubilized by phospholipase C and D varies depending on the age of the culture and presence or absence of fetal calf serum. Different patterns of solubilization were also obtained for 1G7-Ag, depending on whether the test is performed with parasite lysates or with antigen affinity purified from them. Our data indicate that the phospholipase C resistance observed does not arise from acylation on the inositol, as previously described for acetylcholinesterase from human erythrocytes, being rather due to factors which either modify the GPI or affect the action of the phospholipases. Previously unreported resistance to glycosylphosphatidylinositol-specific phospholipase D has been observed both to glycosylphosphatidylinositol-specific phospholipase D has been observed both to 1G7-Ag and 10D8-Ag, the GPI-anchored mucynlike protein which is acceptor of sialic acid in metacyclic forms. Our findings are discussed in the light of the presently known structures of GPI in this parasite, and imaginative speculation on biological roles for the GPI phospholipase system in T. cruzi is also provided.

Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core.

The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.

Glycophosphatidylinositol-anchored proteins in metacyclic trypomastigotes of Trypanosoma cruzi.

We searched for the presence of glycophosphatidylinositol (GPI)-anchored proteins in epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi, by treatment of parasite lysates with the GPI-specific phospholipase C of Trypanosoma brucei. Upon treatment, several proteins (70-90 kDa) in metacyclics, but none in epimastigotes, reacted with antibodies to the cross-reacting determinant (CRD), an epitope revealed on the variant surface glycoproteins of T. brucei following removal of the diacylglycerol moiety from their GPI-anchor. Since these T. cruzi metacyclic proteins also lost their original amphiphilicity, as judged by Triton X-114 phase separation, it is very likely that they are linked to the membrane by GPI. One of these proteins is the 90 kDa protein, the major surface protein of G and Tulahuen strains, recognized by the monoclonal antibody 1G7. A variable portion of the 90 kDa molecules was resistant to solubilization by T. brucei lipase. The reasons for this are not clear but susceptibility appeared to increase with the age of the T. cruzi culture. Enzymes that solubilize GPI-anchored proteins were detected in epimastigotes and metacyclics, but the enzymatic activity in these forms was smaller than the activity detected in the same cell numbers of trypomastigotes of T. cruzi originated from infected mammalian cells or from T. brucei bloodstream forms. A preliminary characterization of these activities indicates that at least two classes of enzymes, one of them inhibited by o-phenanthroline, are present in epimastigotes and metacyclics. None of the reagents tested fully inhibited the phospholipases.