Immunomodulatory activity of glycodelin: implications in allograft rejection.

Glycodelin is an immunomodulator, indispensable for the maintenance of pregnancy in humans. The glycoprotein induces apoptosis in activated CD4+ T cells, monocytes and natural killer (NK) cells, and suppresses the activity of cytotoxic T cells, macrophages and dendritic cells. This study explores the immunosuppressive property of glycodelin for its possible use in preventing graft rejection. Because glycodelin is found only in certain primates, the hypothesis was investigated in an allograft nude mouse model. It is demonstrated that treatment of alloactivated mononuclear cells with glycodelin thwarts graft rejection. Glycodelin decreases the number of activated CD4+ and CD8+ cells and down-regulates the expression of key proteins known to be involved in graft demise such as granzyme-B, eomesodermin (EOMES), interleukin (IL)-2 and proinflammatory cytokines [tumour necrosis factor (TNF)-α and IL-6], resulting in a weakened cell-mediated immune response. Immunosuppressive drugs for treating allograft rejection are associated with severe side effects. Glycodelin, a natural immunomodulator in humans, would be an ideal alternative candidate.

Epitope mapping of Brugia malayi ALT-2 and the development of a multi-epitope vaccine for lymphatic filariasis.

Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.

An evaluation of antigen capture assays for detecting active filarial antigens.

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n= 110), microfilaraemic (MF, n= 65), chronic pathology (CP, n= 45) and non-endemic normal (NEN, n= 10) individuals. Of the 230 samples tested, VAH capture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Differential influence of recombinant non-glycosylated and glycosylated glycodelin on human sperm function: comparative studies with hamster spermatozoa.

Glycodelin, also known as placental protein 14, has been implicated in endometriosis-related infertility. To determine the role of glycodelin and its glycosylated state, the influence of recombinant nonglycosylated-glycodelin (nongly-glycodelin) and glycosylated-glycodelin (gly-glycodelin) on human sperm function was evaluated. Whereas there was a significant (P<0.001) increase in the capacitation of nongly-glycodelin-treated spermatozoa compared with untreated controls (28.8 +/- 1.0% v. 21 +/- 1.5% respectively), treatment of spermatozoa with gly-glycodelin markedly (P<0.001) inhibited capacitation (10.7 +/- 0.3%); acrosome reaction (AR) remained unaltered in all treatments. In a zona-free hamster egg penetration assay, the egg penetration index was higher (P<0.001) with nongly-glycodelin-treated spermatozoa (3.4 +/- 0.3) than with gly-glycodelin-treated spermatozoa (0.4 +/- 0.1) and untreated spermatozoa (1.6 +/- 0.2). A similar influence of glycodelin on capacitation was observed with hamster spermatozoa. However, the AR rate was higher (P<0.01) in nongly-glycodelin-treated spermatozoa (39.4 +/- 1.6%) than in either gly-glycodelin-treated spermatozoa (19.3 +/- 2.0%) or untreated controls (30.0 +/- 1.2%). Moreover, the in vitro fertilization rate was significantly (P<0.01) higher with nongly-glycodelin treated-spermatozoa compared with untreated spermatozoa (77.5 +/- 2.3% v. 52.9 +/- 4.3%) and gly-glycodelin-treated spermatozoa (38.3 +/- 6.5%; P<0.05). These results indicate that whereas nongly-glycodelin improves, gly-glycodelin inhibits, capacitation and fertilization potential of human and hamster spermatozoa, and that the glycosylation status of glycodelin determines its influence on sperm function.

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis.

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.

Helix stabilization in the C-terminal peptide of chicken riboflavin carrier protein enhances immunogenicity and prolongs contraceptive potential as an epitope-based vaccine in female rats.

Earlier investigations have shown that (a) antibodies against a carrier-coupled 20-residue synthetic peptide (C-20), (200)HACQKKLLKFEALQQEEGEE(219), corresponding to the C-terminal partially helical sequence of chicken riboflavin carrier protein (RCP; 219 AA) curtail pregnancy in mammals and (b) helix stabilization by introducing appropriately spaced salt bridges in the flanking sequences of its B-cell epitopic structure enhances RCP antigenicity to peptide antibodies. Among such engineered C-20 analogs, HE-20 (HAEQKKLLKFEALEQEKGKE) exhibited maximum helical propensity. Since C-20 per se, i.e., without carrier conjugation, elicits RCP-reactive neutralizing antibodies in rodents, we mapped its T-cell epitope which overlaps its B-cell epitope, both of which remain unmodified in HE-20. Comparative evaluation of immunogenicity of the two epitope-based peptide vaccines showed that HE-20 was far superior to C-20 in generating RCP-reactive antibodies in terms of both affinity and titer. With regard to bioefficacy, passive immunoneutralization of RCP in pregnant rats by administering purified IgG from either of the antipeptide sera terminated pregnancy. Similarly, active immunization of fertile female rats with the individual peptide analogs curtailed pregnancy. However, HE-20 was more efficient in eliciting higher affinity, longer-lasting, RCP-crossreactive antibodies with consequently more prolonged immunocontraceptive efficacy.

Riboflavin carrier protein: a serum and tissue marker for breast carcinoma.

We have earlier shown that the estrogen-modulated riboflavin carrier protein (RCP) first isolated from the chicken egg is evolutionarily conserved in mammals and is elaborated by lactating mammary gland as demonstrated with rat mammary epithelial cells in culture and confirmed by isolation of the vitamin carrier from bovine milk. In view of several earlier reports that many milk proteins as well as other estrogen-inducible proteins are up-regulated and secreted into circulation in animal models and in women with neoplastic breast disease, we analyzed serum RCP levels in a double-blind study using a specific radioimmunoassay in pre- and post-menopausal women with clinically diagnosed breast cancer at early and advanced stages of the disease and compared these levels with those in normal age-matched control volunteers. Our data reveal that the serum RCP levels in cycling breast cancer patients are 3- to 4-fold higher (p < 0.01) than those in their normal counterparts. This difference in circulatory RCP levels between cancer patients and their age-matched normal counterparts is further magnified to 9- to 11-fold (p < 0.005) at the post-menopausal stage. In addition, there seems to be a good correlation between rising RCP levels and disease progression, since significantly higher RCP concentrations (p < 0.005) are encountered in patients with advanced metastasizing breast cancer versus those with early disease. Using specific monoclonal antibodies, RCP could be localized immunohistochemically in the cytoplasm of invading neoplastic cells of lobular and ductal carcinomas of the breast, indicating that the malignant cells are probably the source of the elevated serum RCP levels in breast cancer. These findings suggest that measurement of circulatory RCP and the immunohistochemical staining pattern of RCP in biopsy specimens could be exploited as an additional marker in diagnosis/prognosis of breast cancer in women.

Placental protein 14 induces apoptosis in T cells but not in monocytes.

Substantial evidence exists in literature to suggest that placental protein 14 (PP14) (recently renamed glycodelin A), exhibits immunosuppressive properties and is an indispensable macromolecule in the maternal system for the establishment, maintenance, and progression of pregnancy. Though there are several reports substantiating the above, the mechanism of its action at the molecular level has not been elucidated as yet. In this paper we provide data that suggest that amniotic fluid PP14 and recombinant PP14 expressed in Pichia pastoris induce apoptosis in human peripheral blood lymphocytes upon activation, independent of monocytes. That PP14 has a direct apoptotic action on T cells but not on monocytes was also demonstrated by utilizing human cell lines. PP14 was shown to induce apoptosis in the human T cell lines, Jurkat and MOLT-4 cells, but not in the human monocytic cell line, U937.

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast.

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.

Immunocontraceptive efficacy of synthetic peptides corresponding to major antigenic determinants of chicken riboflavin carrier protein in the female rats.

PROBLEM: Earlier studies have demonstrated that antibodies directed towards the N-terminal (residues 10-17) and C-terminal (residues 200-207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub-human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33-49, 64 83 and 130-147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated. METHOD OF STUDY: The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies. RESULTS: All the three peptides-specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE-IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy. CONCLUSIONS: These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini-vaccines.

Immunocontraceptive potential of major antigenic determinants of chicken riboflavin carrier protein in the female rat.

Earlier investigations have demonstrated that antibodies generated against the N-terminal (10-17) and C-terminal (200-207) ends of chicken riboflavin carrier protein (RCP; 219 AA), but not towards the internal region (172-179), curtail pregnancy establishment in rodents and sub-human primates. In those studies, epitope peptides conjugated to diphtheria toxoid were used as immunogens. In the present study, linkage of these sequences to extraneous carriers was avoided to rule out the possibility of carrier-mediated suppression of hapten-specific antibody production in long-term immune response. The ability of these three free peptides to function as minivaccines was examined and the functional importance of these sequences in pregnancy establishment in rodents were evaluated. The results obtained reveal that the peptides YGC (residues 3-24) and HAC (residues 200-219) serve as immunocontraceptive vaccines.

Characterization of monoclonal antibodies to glycodelin and recombinant glycodelin.

Glycodelin-A belongs to the lipocalin superfamily. Although it is associated with normal endometrial growth during the menstrual cycle, fertilization and normal pregnancy in humans, the molecular mechanism of its biological action has not been elucidated. To undertake studies to understand the functional relevance of any molecule, obtaining large quantities of the protein becomes essential. With the ultimate aim of purifying glycodelin either from its natural sources (human amniotic fluid) or the recombinant glycodelin from bacterial recombinant lysates, we raised monoclonal antibodies to this protein. As immunogens, recombinant glycodelin expressed in E. coli and Pichia pastoris as well as glycodelin from amniotic fluid were used. The monoclonal antibodies generated were characterized with respect to binding to both the native as well as the recombinant proteins using ELISA, immunoblotting, and immunohistochemistry.

Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and a conjugate of ricin B-chain with momordin.

Ribosome inactivating proteins from plants have been widely used for the preparation of immunotoxins and hormonotoxins, which have potential application in the therapy of diseases such as cancer. However, these hybrid toxins have been found to be less cytotoxic than native ribosome inactivating proteins. Therefore, it is important to understand the factors that control the intrinsic toxicity of RIPs and the hybrid toxins prepared using them. Here, a hybrid toxin has been prepared by coupling ricin B-chain to momordin and the cytotoxicity of this hybrid toxin has been compared to that observed in case of native ricin. In the two cell types used here, thymocytes and macrophages, the conjugate was found to be about 40 fold less toxic than native ricin. Kinetics of inhibition of protein synthesis showed that prior to onset of inhibition the conjugate exhibits a longer lag phase than native ricin. The rates of inhibition of protein synthesis by the conjugate were also found to be slower than ricin. Analysis of the results suggests that in addition to cell surface binding, the B-chain of ricin facilitates another step in the transmembrane translocation of ricin A-chain to the cytosol.

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Molecular mimicry by antiidiotypic monoclonal antibody to gonadotropin releasing hormone.

Antipeptide and antiidiotypic antibodies to several receptors are known to mimic their respective ligands in transducing signals on binding their receptors. In our attempts to study gonadotropin releasing hormone receptor, antipeptide and antiidiotypic monoclonal antibodies specific to the receptor were established earlier. The antipeptide mAb F1G4 was to a synthetic peptide corresponding to the extracellular domain of human GnRH receptor and the antiidiotypic mAb 4D10C1 was to the idiotype of a GnRH specific mAb. Here we report the physiological effects of the two mAbs on binding the receptor, as investigated using in vitro cultures of (a) human term placental villi and (b) rat pituitaries. The mAb 4D10C1 exerted a dose-dependent release of human chorionic gonadotropin in cultures of human term placental villi as well as luteinising and follicle stimulating hormones in cultures of rat pituitaries.

A comparison of two dissimilar monoclonal antibodies that are directed to a common epitope in the reduced and carboxymethylated chicken riboflavin carrier protein.

Two monoclonal antibodies, D2A1 and D5G3 were elicited by immunization with a preparation of chicken egg riboflavin carrier protein which had been reduced and carboxymethylated. Epitopes recognised by the monoclonal antibodies were mapped using the Pepscan method. Epitopic determinants for D2A1 as well as D5G3 were identified within a region spanning 13 amino acids (residues 170-182) in the primary sequence of riboflavin carrier protein. Interestingly, these monoclonal antibodies, despite sharing a common epitope exhibited a marked difference in their binding to native (folded) riboflavin carrier protein versus reduced carboxymethylated (unfolded) riboflavin carrier protein. Both monoclonal antibodies bound reduced carboxymethylated riboflavin carrier protein to comparable extents in solid phase (ELISA and immunoblots) and liquid phase (radioimmunoassay) assays. However, while D5G3 could bind native riboflavin carrier protein in solid and liquid phase assays, D2A1 showed negligible binding to the native structure. Alanine substituted peptide analogs of the epitope in question defined the crucial amino acids of the epitope needed for binding to the two antibodies.

Antiidiotypic antibody to gonadotropin releasing hormone as probe for cell surface receptors.

Monoclonal antibodies were raised to the idiotype of a gonadotropin releasing hormone (GnRH) specific antibody, one of which was found to bind specifically to GnRH receptors present on pituitary gonadotrophs, placental syncytiotrophoblasts and testicular Leydig cells. These observations were confirmed by Western and ligand blotting as well as by the ability of the antibody to induce an increase in intracellular Ca++ in a mouse gonadotroph cell-line viz., alpha T3-1.

Placental protein 14 in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium in the rhesus monkey.

Placental protein 14 (PP14) is a glycoprotein which is secreted by secretory phase endometrium and decidua in women. Despite the suggestion that PP14 is involved in the process of endometrial maturation for blastocyst implantation, our understanding in this regard is poor. In the present study, the concentrations and distribution patterns of immunodetectable PP14 in the endometrium during proliferative and secretory phases of normal ovulatory menstrual cycles, as well as in implantation stage endometrium in naturally mated ovulatory cycles with or without early luteal phase mifepristone treatment, were investigated using the rhesus monkey as a primate model. Immunopositive PP14 was observed mainly in epithelial cells of glands and it was detected in one major immunopositive band at Mr 28 kDa in tissue homogenate and spent medium. The area of immunopositive precipitation of PP14 in glands was minimal in follicular phase endometrium, and was higher (P < 0.01) in early, mid- and late luteal phase endometrium compared with that in pre- and periovulatory phases of the cycle, but there was no change in its area profile in the glandular compartment throughout the luteal phase. Immunopositivity for PP14 in luminal contents of gland displayed an increasing profile from early to late secretory phases. Thus, the concentrations and the distribution of immunodetectable PP14 in luteal phase endometrium of the rhesus monkey showed marked similarity with those of human endometrium during the natural menstrual cycle. Although there was no marked change in the band characterstics for the protein in implantation stage endometrium following early luteal phase mifepristone treatment, it was markedly decreased (P < 0.01) in tissue homogenate and in vitro spent medium along with a lesser (P < 0.02) degree of immunoprecipitation in the glands in implantation stage samples of mifepristone treatment group compared with that in control group samples. Thus, the contragestional effect of early luteal phase mifepristone treatment appears to be associated with a decrease in the concentration of immunodetectable PP14 in implantation stage endometrial glands and its secretion in the rhesus monkey. It remains to be seen whether this decline is caused from direct antiprogesterone action on endometrial glands during progesterone dominance, or secondarily from associated retarded development of endometrium.

Cloning, expression, purification, and immunocharacterization of placental protein-14.

Human placental protein-14 (PP-14), a member of the lipocalin superfamily, shares homology at the level of the primary and secondary structures with bovine beta-lactoglobulin. It is the most prominent endometrial protein synthesized by the glandular cells of endometrium under estrogen priming and progesterone stimulation. The temporal and spatial expression of PP-14 in the female reproductive tract combined with its biological activities ex vivo suggest that this glycoprotein probably plays an essential physiological role in the regulation of fertilization, implantation, and maintenance of pregnancy. We proposed to elucidate the molecular mechanisms involved in the function of this protein. A prerequisite to such investigations on any protein is the availability of sufficient amounts of the same in a homogenous form. Therefore, recombinant DNA technology was employed. The PP-14 cDNA was obtained from the first-trimester endometrial tissue RNA by RT-PCR using unique primers. After confirming the identity of the gene, the protein was expressed in Escherichia coli and purified to homogeneity. The gene was also cloned and expressed in Pichia pastoris to obtain the protein product in a glycosylated form. The recombinant proteins were immunocharacterized using a cross-reactive antibody raised to bovine beta-lactoglobulin. Polyclonal antiserum raised to the E coli expressed PP-14 also bound to the native PP-14 from amniotic fluid suggesting that recombinant PP-14 may be exploited to elucidate functional aspects of the protein.

A monoclonal antibody to avidin dissociates quaternary structure and curtails biotin binding to avidin and streptavidin.

An anti-avidin mAb, viz., H12G4, is shown to release bound biotin in a dose-dependent manner from holoavidin and holostreptavidin and inhibit the binding of ligand to the two apoproteins. The release of biotin by this mAb is accompanied by quenching of ligand-induced enhanced fluorescence of the FITC-avidin conjugate. In terms of mechanism of release of bound biotin, we demonstrate that on binding to the Fab fragment of the mAb, the native tetrameric holoavidin undergoes dissociation progressively with time to monomers with no bound biotin associated with the latter. Based on the immunoreactivity associated with defined overlapping fragments of avidin obtained by chemical cleavage, the epitope recognized by mAb H12G4 has been localized to residues 58-96 of the primary sequence. By pepscan method of epitope mapping, this mAb is shown to identify a minimal core sequence of 87RNGK90 in avidin and a corresponding sequence of 84RNAH87 in streptavidin.