Identificación y caracterización del componente proteico de la especialidad farmacéutica Inmunoferon®

Abstract

INTRODUCTION The immune system is the body's defence system involved in protection against micro-organisms, pathogens and neoplasia. This system is comprised of a large variety of cells and molecules able to specifically recognise molecular structures or antigens and carry out an immune response that leads to their removal. However, at times this response may be altered leading to diseases arising from insufficient response (immunodeficiency, infection, and neoplasia) or excessive response (allergy, autoimmunity, transplant rejection). The therapeutic strategy used to restore correct operation of the immune response, either by stimulating or suppressing it, is known as immunomodulation. Highly varied immunomodulator agents are used to attain immunomodulation; these include synthetic, recombinant and natural origin substances. Within the latter group immunomodulators designed with the aim of stimulating natural immunity mechanisms are notable. The Spanish drug Inmunoferon® belongs to this group. This is an oral immunomodulator which has proved its ability to normalise the effector function of accessory and phagocytic cells, natural killer cells and T lymphocytes. At the same time it inhibits the production of TNF-α and modulates the production of other regulatory cytokines (IL-1, IL-2, IL-12, IFN- γ ). It has been used in various diseases such as chronic hepatitis B, chronic obstructive pulmonary disease, apthous stomatitis and muscular inflammation, among others. The active substance of the specialty Inmunoferon® is a non-covalent association of polysaccharide/protein absorbed on a stabilising matrix of calcium sulphate and phosphate. The polysaccharide is a glucomannan from the Candida utilis wall and the protein component arises from non-germinated ricin seeds (Ricinus communis). To date, the total lack of knowledge over the nature of this protein component has hindered studying and ascertaining in depth the complex pharmacology of this drug.

OBJECTIVES 1- To obtain the different polypeptides comprising the organic component (AM5) of the active substance (AM3) of the pharmaceutical specialty Inmunoferon® to perform its molecular identification and characterisation. 2- Structural analysis of secondary structure by means of the circular dichroism technique of polypeptides isolated from AM5. This analysis will be completed with a study of the conformational stability of these polypeptides by performing denaturalisation experiments induced by chemical agents, temperature and pH. 3- Stability study compared to the most important proteins of the gastrointestinal tract. 4- Obtaining preparations of protein marked with 15N, with suitable homogeneity to elucidate their three-dimensional structure at high resolution. With this purpose in mind the DNA sequence coding for the majority polypeptides present in AM5 will be designed; a system of expression that enables obtaining sufficient amount of these isotopically marked proteins will be carried out. RESULTS Analysis of the organic component of Inmunoferon® by SDS-PAGE revealed the presence of three unique bands with apparent molecular mass ~20, 7.4 and 5.2 kDa. These three polypeptides, which we have called AM55, AM56 and AM57, were purified to homogeneity by means of a gel filtration chromatography stage followed by reverse phase column chromatography. Analysis by SDS-PAGE in the presence and absence of reducer agents revealed that AM55 and AM56 are heterodimers comprised of two subunits with size 6.2 and 4.6 kDa, and 5.2 and 3 kDa, respectively, bound by disulphide bridges, whilst AM57 is comprised of a single polypeptide chain with mass coinciding with that of the highest weight subunit of AM56. AM55, AM56 and AM57 were analysed by means of techniques for analysing amino acids, terminal amino sequencing and mass spectrometry; AM55 was identified as the reserve protein of ricin seeds (Ricinus communis) Ric c 3 (da Silva Jr. et al., 1996, Bashir et al., 1998). AM56 was also identified as the reserve protein of these seeds Ric c 1 (AM56, Sharief & Li 1982, Bashir et al., 1998). These proteins belong to the family of albumins 2S and proceed from a common precursor polypeptide of 34 kDa whose proteolytic processing leads to mature proteins (Irwin et al., 1990). Ric c3 is constituted by two chains of molecular masses 4 and 7 kDa, bound together by two disulphide bridges; there are also another two intracatenary bridges in the heavy chain. AM57 is exclusively constituted by the largest subunit of Ric c1. Detailed analysis of the mass spectra of AM55, AM56 and their corresponding subunits, previously separated with DTT and purified by means of reverse phase chromatography, revealed the presence of some heterogeneity. We have been able to establish that the origin of this heterogeneity lies in the non-specific processing of the precursor polypeptide, specifically in the two segments that connect the chains, which in the case of AM56 produces protein forms with 1 or 2 additional amino acids, or 1 amino acid less on the carboxyl end of the light chain; in AM55 this gives rise to a form with 4 amino acids more on the carboxyl end of the light chain and another with 1 amino acid less on the carboxyl end of the heavy chain.