Studies on vitamin carrier proteins: physicochemical and functional aspects

Abstract

STUDIES ON VITAMIN CARRIER PROTEINS: PHYSICOCHEMICAL AND FUNCTIONAL ASPECTS submitted by P.B. SESHAGIRI for the award of Ph.D. degree of the Indian Institute of Science, Bangalore, India. Investigations detailed in this thesis constitute a part of the continuing programme of research work in this laboratory on vitamin carrier proteins from avian and mammalian species (including primates) with particular reference to their isolation, characterization, and functional significance during embryonic development. This thesis is divided into two parts. Part A describes the isolation and characterization of biotin-binding protein (BBP) from the chicken egg white distinguishable from avidin (Chapter I) and of a similar protein in the sera of pregnant or estrogen (E) treated rats and its functional significance during embryonic development (Chapter II). Part B includes the experiments dealing with active immunization against chicken riboflavin carrier protein (cRCP) of female bonnet monkeys (Macaca radiata) and the consequence thereof in terms of maintenance and progression of pregnancy. PART A: Although the essential requirement of vitamins like biotin for the embryonic development in the chicken has been known for a long time, the mode of their protein-mediated deposition in the egg is exemplified by the discovery of specific high-affinity vitamin-binding/carrier proteins. Some of these are present both in the yolk and the white compartments of the egg and they share several physicochemical and immunological properties. The only apparent exception to this is the egg yolk BBP which is physicochemically and immunologically distinct from avidin of the egg white. In order to explore the possibility of the existence of another BBP in the egg white, distinguishable from avidin, yet exhibiting several features in common with the yolk BBP, a search was carried out on these lines. The data of Chapter I clearly provide immunological and biochemical evidence for the existence of BBP distinct from avidin in the egg white which culminated in its isolation, characterization, and comparison of its properties with those of the yolk BBP and avidin. Ouchterlony immunodouble diffusion technique revealed that the oviduct cytosols of the estrogenised chicks/laying hens and the crude protein fraction of the egg white contained BBP that exhibited immunological cross-reactivity with the purified yolk BBP. The cross-reacting protein could be quantitatively immunoprecipitated and sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed that the protein has a molecular weight (Mr) of 67,000 without any detectable subunits. This protein of the egg white exhibited specific biotin-binding activity and when saturated with the unlabelled biotin, the protein displayed thermally induced biotin exchange reaction at 55°C. Analysis of in vitro biosynthesised labelled egg white proteins in the oviduct tissue explants from estrogenised chicks revealed that ~2% of the total labelled proteins could be specifically immunoprecipitated with anti-yolk BBP antibodies. These interesting findings led to the isolation of BBP from the egg white by employing DEAE-cellulose chromatography and biotin-affinity chromatography. The isolated protein was electrophoretically homogeneous, specifically bound biotin, had an Mr of 67,000 and exhibited complete immunological cross-reactivity with the yolk BBP and yet was physicochemically and immunologically distinct from avidin. Moreover, several physicochemical characteristics of this protein were similar to those of the yolk BBP, which include acidic nature, native Mr, biotin-binding characteristics, immunological cross-reactivity, and E-requirement for biosynthesis. The tryptic peptide map of the white BBP was comparable to that of yolk BBP, but differed from that of avidin. In vitro translation of poly(A)+ RNA from the liver and oviduct and the Western blot analyses of the cytosols of the liver and oviduct indicated that in the liver, BBP is synthesised as a large polypeptide of size corresponding to native Mr of white BBP but gets cleaved to form identical subunits by specific, selective proteolytic processing at the level of ovarian uptake, whereas the oviduct synthesises a 67,000 BBP which gets deposited in the egg white as such. Earlier, specific carrier proteins for riboflavin and thiamin have been isolated in our laboratory from the pregnant rat serum and were shown to exhibit immunological cross-reactivity with their respective avian counterparts. They were E-inducible proteins of hepatic origin and were indispensable in transplacental vitamin transport. Developed for this protein by using yolk BBP-antibodies. However, the kinetics of hormonal induction of this protein in the rat could not be investigated, in view of the remarkable immunological cross-reactivity between rBBP and the rat serum albumin (RSA). Other gross immunochemical methods however failed to show this cross-reactivity. However, the pI of rBBP differed from RSA. This was confirmed by analysis of a mixture of radioiodinated BBP and RSA by non-equilibrium pH gradient acrylamide gel electrophoresis which revealed different mobilities in the first dimension (IEP) but identical mobility in the second dimension (SDS-PAGE). These significant differences in the physicochemical properties of the two proteins were further confirmed by comparing the tryptic peptide maps of the iodinated BBP and RSA. These findings revealed that rBBP and RSA are immunologically (RIA) similar but they do exhibit several clear-cut differences in terms of their physicochemical properties. Another approach to differentiate between rBBP and RSA is to probe into the functional importance of rBBP during embryonic development by bringing about selective immunoneutralisation of the endogenous BBP by eliciting anti-yolk BBP antibodies in the rats by active immunisation. The immunised rats experienced repeated early pregnancy termination as revealed by laparotomy on day 18 of expected pregnancy and the sharp decline in maternal plasma progesterone (P) by day 8 of pregnancy. This phenomenon of pregnancy termination consequent on active immunisation could be demonstrated as long as the antibody titers were maintained by periodic boosters with the antigen. Immunisation per se had no effect on the status of general health, weight gain, reproductive cyclicity and gross biotin status of the animals. Thus these experiments have revealed that maternal BBP is essential for embryonic development and presumably plays a vital role in terms of biotin supply to the fetus. PART B: Earlier results obtained in this laboratory have revealed that immunoneutralisation of RCP in the pregnant rats terminates their early pregnancies as a consequence of curtailment of riboflavin supply to the developing fetuses, culminating in acute fetal wastage and hence pregnancy termination. An extension of such studies to the non-human primate model viz., bonnet monkey resulted in the discovery of RCP in this animal during pregnancy. The monkey RCP (mRCP) has been isolated to apparent homogeneity and most of its physicochemical properties are comparable to those of the cRCP. In order to study the functional significance of mRCP during embryonic development of the monkey and to demonstrate whether the phenomenon of repeated pregnancy termination following active immunisation against chicken RCP observed in the rodents is also operative in the sub-human primate, five female bonnet monkeys of proven fertility were actively immunised against cRCP. Part B of this thesis embodies the results obtained on these lines. All the animals elicited good immunological response, although the immunopotencies of the sera were varying, a definite fraction of antibodies to cRCP recognised mRCP, indicating the presence of a small sub-population of circulating antibodies which could immunoneutralise the endogenous RCP. Immunisation per se had no effect on the status of general health, menstrual cyclicity, reproductive hormonal profiles and the riboflavin status of these animals. The pregnancies in the actively immunised animals were monitored by estimating the levels of progesterone and monkey chorionic gonadotropin (mCG). Results obtained over a period of 2½ years indicated that there is immunosuppression of pregnancy in at least 4 out of 5 animals at various times. Pregnancy termination (abortion) occurred between days 12 and 61 after conception as revealed by the declining pattern of pregnancy-specific hormones and profuse vaginal bleeding. Abortion appears to depend on the titre of immunoneutralisable antibodies in circulation. Those animals that escaped pregnancy termination concordantly showed low titres of cRCP antibodies as gauged by radioimmunochemical and quantitative immunoprecipitation analyses. The animals that had showed pregnancy termination once or more after repeated exposure to males eventually carried the pregnancy to term and delivered either stillborn or live babies. The offsprings did not show any visible teratological abnormalities. This escape from pregnancy termination is presumed to be due to acquisition of immune tolerance over a period of time, upon repeated booster injections with the antigen. Although unlike in the rodent model, in the primates 100% pregnancy termination due to active immunisation against cRCP could not be demonstrated, the protein-mediated vitamin delivery mechanism appears to be definitely operative. Furthermore, these studies widen the scope of research on female immunocontraceptive methods, since one more potential antigen, viz., cRCP, can be exploited in future.