Significance of hydrophobic and charged sequence similarities in sodium-bile acid cotransporter and vitamin D-binding protein macrophage activating factor

Ibne Raihan Zunaid; Stefania Pacini; Marco Ruggiero

The sodium-bile acid cotransporter, also defined the sodium-taurocholate cotransporting polypeptide (NTCP) or also liver bile acid transporter, is an integral transmembrane protein that is encoded by the SLC10A1 (solute carrier family 10, member 1) human gene and shows multiple hydrophobic transmembrane domains (1) . This transporter plays a critical role in maintenance of the enterohepatic recirculation of bile acids and hepatocyte function. The protein is constituted by 349 aminoacids with a resulting molecular mass of 56 kDa and works by binding two sodium ions and one (conjugated) bile salt molecule, in this manner determining hepatic influx of bile salts. Other molecules that may be transported by this protein comprise steroid and thyroid hormones, and xenobiotics of different origin (2) . NTCP also represents the receptor for Hepatitis B virus (HBV) and the interaction between the aminoacid sequences of NTCP and those of HBV surface proteins has been characterized in molecular details. The preS1 sequence of large envelope protein (L) is essential for interaction with a stretch of nine hydrophobic residues (157-KGIVISLVL-165) located in the third transmembrane domain of NTCP. The sequence of the preS1 L protein binding NTCP is constituted by a stretch of predominantly hydrophobic aminoacids (residues 2-48) and, within this stretch, there is a highly conserved motif (9-NPLGF(F/L)P-15) that is essential for binding (3) . It is apparent that knowledge of the mechanics of interaction at this level may be instrumental in developing strategies aimed at preventing preS1/NTCP interaction with consequent internalization. We therefore decided to use computational tools to investigate sequence similarities in other proteins that may interact with the preS1 sequence as well as with other surface proteins of viruses. To this end, we chose a well-characterized multifunctional serum protein that has the ability to work as a transporter, a hormone-and fatty acid binding protein, and an immune stimulating cytokine, the vitamin D-binding protein (DBP) macrophage activating factor (MAF). This is a serum alpha-2 glycoprotein constituted by a single polypeptide chain showing a molecular mass of 51-58 kDa. From the structural point of view, it is related to serum albumin and it is also known as Gc (Group-component) globulin. DBP synthesis occurs in the liver and its level in human plasma is around 20-55 mg/100 ml. DBP is a multifunctional protein that, in addition to vitamin D, binds actin, acting as an actin scavenger, and also binds fatty acids. The protein is constituted by 458 aminoacids and shows three domains that share limited sequence similarity with each other as well as with similar sequences of human albumin. The first domain contains the vitamin D-binding site that is located in a shallow cleft that allows interaction with the plasma membrane. DBP is present on the surface of several cell types that comprise yolk sac endodermal cells and T lymphocytes. In B lymphocytes, DBP is responsible for the linkage of surface immunoglobulins, thus contributing to the balance of the immune response (4). The de-glycosylated form of DBP is a powerful macrophage activating factor -hence the designation DBP-MAF -and shows a number of biological effects (5) . We demonstrated that, in addition to its presence in human serum, DBP-MAF is formed during fermentation of milk and colostrum (6) as well as during fermentation of hemp seed proteins (7). Here, we demon