Studies on thiamin carrier proteins : physicochemical, biosynthetic and functional aspects

Abstract

Synopsis of the thesis entitled "STUDIES ON THIAMIN CARRIER PROTEINS (TCP): PHYSICOCHEMICAL, BIOSYNTHETIC AND FUNCTIONAL ASPECTS" submitted by P. V. MALATHY for the award of Ph.D. degree of the Indian Institute of Science, Bangalore, India. Earlier investigations in our laboratory have demonstrated for the first time that thiamin deposition in the chicken egg is accomplished by a specific carrier protein–mediated process. As part of the continuing programme on the molecular and physiological significance of vitamin carrier proteins, studies were undertaken to investigate the possibility of participation of a similar carrier mediated thiamin delivery mechanism during gestation in mammalian species to subserve fetal vitamin nutrition. This study was prompted by observations made elsewhere that despite the placental membrane offering a barrier to the transport of vitamins and their coenzymes, the fetal tissue has a remarkable capacity to accumulate these micronutrients from the maternal circulation even under conditions of hypovitaminemia. The major part of the work described in this thesis (Chapters I, II and III) was carried out in a lower mammal, viz., the rat, as the model system. A specific high affinity TCP was identified and isolated for the first time from pregnant or estrogenised rat sera by affinity chromatography on TPP AH Sepharose. The isolated protein (rTCP) was homogeneous and biologically active in terms of its specific high affinity interaction with ¹ C thiamin. rTCP is a glycoprotein of 55,000 Da, devoid of subunits, and with an affinity constant for thiamin of the order of 4 × 10 M ¹. Interestingly, rat TCP exhibited lower affinities for the phosphorylated derivatives of thiamin, viz., TMP and TPP, which is suggestive of the involvement of TCP in the physiological transport of thiamin only and not its esters. The purified protein showed partial immunological cross reactivity with its chicken counterpart, as detected by immunodouble diffusion. To understand the biosynthesis and endocrine regulation of rTCP, a specific and sensitive RIA was developed. This assay procedure was employed to assess the hormonal modulation of TCP during the normal reproductive phases of the animal as well as during experimental estrogenisation. The physiological modulation of TCP was examined by monitoring the levels of the vitamin carrier during estrous cycle and pregnancy with a view to ascertain a possible correlation of its plasma levels with the concurrent changes in circulatory estrogen concentration. It was observed that TCP levels fluctuated in concert with changing estrogen levels during the estrous cycle and early pregnancy. A strict temporal relationship between TCP and estrogen concentration was not evident during the later stages of pregnancy, presumably because of the occurrence of multihormonal interactions and/or the “sink effect” exerted by the fetoplacental unit. To investigate the kinetics of estrogen induced elaboration of TCP, immature male rats were primed with a single dose of estradiol 17 and the plasma levels of TCP monitored by RIA. Following injection of the steroid hormone, TCP rapidly accumulated in plasma, attaining peak concentrations at 48 h, and declined thereafter. A one and a half fold amplification of the inductive response was observed following secondary stimulation with the hormone. This phenomenon is analogous to the “memory effect” observed with vitellogenin, riboflavin carrier protein, and biotin carrier protein in immature chicks primed with the steroid hormone. The magnitude of the inductive response exhibited a clear dependency on the dose of the steroid hormone. Besides inducing the de novo synthesis of TCP, estrogen also exerted a stabilising effect on TCP catabolism, as was evident by a doubling of the circulatory half life of TCP under conditions of estrogenisation. The inductive effect of estrogen was severely curtailed in the presence of antiestrogens, while progesterone was incapable of either modulating the estrogen induced response or eliciting an inductive response by itself. Further, cycloheximide drastically blocked the response to estrogen, which was indicative of de novo synthesis. Thus, it appears that the elaboration of TCP is strictly under estrogenic influence. Studies were undertaken to assess the TCP synthesising capacity of yet another target organ for estrogen, viz., the uterus, besides the liver. Conclusive proof for the synthetic capacity of the uterus was obtained by in vitro incorporation of radioactive amino acids into uterine tissue explants of estrogenised female rats. The elaboration of TCP by these two target organs, liver and uterus, exhibited several similarities in terms of hormonal specificity, kinetics of induction, and modulatory aspects. TCP levels in uterine luminal fluid were also measured during estrous cycle, pregnancy, and experimental estrogenisation. The uterine content of TCP showed a strict dependency on hormonal dose, analogous to that observed in plasma. The finding that rodent liver and uterus serve as two independent sites of TCP synthesis emphasises the evolutionary conservation of TCP not only in terms of structural and functional features as a vitamin carrier, but also with regard to the dual sites of synthesis-liver and oviduct/uterus-during the reproductive phase, as well as the hormonal principles involved in induction. Immunological approaches were adopted to establish the functional importance of TCP during gestation. Immunoneutralisation of circulating maternal TCP was effected by both passive and active immunisation experiments. Passive immunoneutralisation with either heterologous or homologous antibodies to TCP resulted in a drastic curtailment of ¹ C thiamin influx into embryos, with a concomitant decrease in embryonic weight. In contrast, embryos from non immunised mothers showed a steady, linear increase in the uptake of the vitamin with time, accompanied by a concomitant increase in embryonic weight. It was observed that multiple doses of anti TCP IgG were essential to precipitate 100% fetal resorption. Examination of intraembryonic events leading to acute fetal wastage/death revealed gross alterations in thiamin metabolism selectively in fetal tissue, since uptake and metabolism of the vitamin in maternal liver were unaffected. In embryos from control animals (non immune rabbit serum–administered), more than 50% of the radioactivity was located in the TPP fraction; whereas in affected embryos, only 25% of absorbed radioactivity was associated with TPP, the major portion being predominantly associated with unphosphorylated thiamin. These results suggest that the molecular mechanism leading to acute fetal resorption/death involves drastic curtailment of thiamin supply to embryos, with concomitant drastic changes in fetal thiamin metabolism. These findings raised the possibility of employing active immunisation with heterologous protein, viz., chicken TCP (cTCP), as an effective tool for suppression of pregnancy in the rat. Active immunisation of fertile female rats with cTCP led to early termination of pregnancy, with no effect on implantation per se. Pregnancy termination could be observed as long as antibody titres were maintained at high levels by booster doses of the antigen. The sudden and precipitous fall in circulatory progesterone and estrogen concentrations was indicative of fetal distress, which subsequently culminated in acute fetal wastage. When these animals were allowed a rest period in which no booster shots were administered, they conceived and delivered normal pups. Active immunisation produced no discernible adverse effect on the mothers with respect to cyclicity, thiamin status, or subsequent reproductive capacity. To confirm the results of passive immunisation-that fetal wastage was due to drastic curtailment of thiamin supply-massive doses of thiamin were administered to pregnant cTCP immunised animals under conditions of high antibody titres. The affected embryos could indeed be rescued by such vitamin therapy. Since evolutionary conservation of immunological and functional characteristics existed between avian and rodent TCPs, attempts were made to examine the existence of a similar protein during pregnancy in higher mammals, including primates. TCP was found to be highly conserved throughout evolution, as evidenced by proteins exhibiting immunological cross reactivity with cTCP and rTCP antibodies. Using the same bioaffinity chromatography procedure, a high affinity TCP was isolated from pooled human pregnancy serum. The isolated protein was homogeneous by PAGE; it was a glycoprotein with M 60,000. The purified protein (hTCP) exhibited immunological similarity with cTCP and rTCP, and interacted with thiamin with high affinity. A similar protein was purified from human umbilical cord serum. TCP purified from cord and maternal sera were similar with respect to glycoprotein nature, vitamin binding properties, and immunological cross reactivity. These findings provide conclusive evidence for the functional importance of TCP in fetal vitamin nutrition. The fact that TCP is conserved through different stages of evolutionary transition signifies the vital role played by this vitamin carrier in embryonic development. It therefore appears that for embryonic nutrition and survival, an adequate supply of vitamin has been ensured by adequate amounts of maternal vitamin carrier; any disturbance in the production or functioning of these proteins could have grave consequences on fetal health and well being.