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

Study of sequence similarities were performed using the Align tool of Uniprot (uniprot.org). For NTCP, the sequence UniProtKB -Q14973 (NTCP_HUMAN) was used. For DBP-MAF, the sequence UniProtKB -P02774 (VTDB_HUMAN) was used. For HBSAG, the sequence UniProtKB -P03138 (HBSAG_HBVD3. Isolate France/Tiollais/1979) was used. For alpha-Nacetylgalactosaminidase (abbreviated in NAGAB_HUMAN), the sequence UniProtKB -P17050 (NAGAB_HUMAN) was used. The sequences TNGTKR, HKNNKS, RSYLTPGDSSSG, and QTNSPRRA were retrieved from Jaimes et al. (10) . The yellow color used in Fig 1 indicates the transmembrane domains of NTCP. The gray color indicates similarities; the conventional consensus symbols are: "*" indicating that the residues are identical in all sequences in the alignment. ":" indicating that conserved substitutions have been observed. "." indicating that semi-conserved substitutions are observed, that is, amino acids having similar features. The light indigo color indicates hydrophobicity. The pale red color indicates negatively charged residues. Study of in vitro interaction between DBP-MAF or the product of microbial fermentation of milk and colostrum mentioned above, here abbreviated in PMF (Product of Microbial Fermentation), and NAGAB_HUMAN was was performed by R.E.D. Laboratories (Zellik, Belgium) where NAGAB_HUMAN and DBP-MAF had been purified. NAGAB_HUMAN is an enzyme that cleaves terminal alpha-N-acetylgalactosamine residues from glycolipids, glycopeptides and glycoproteins; it binds to the alpha-N-acetylgalactosamine moiety that is attached to threonine in the sequence TPTELAK close to the carboxyl terminus of DBP-MAF. Therefore, binding of NAGAB_HUMAN is a direct method to evaluate DBP-MAF activity in serum or other matrices. For these experiments, microtiter plates coated with a specific antibody directed against NAGAB_HUMAN were used. Samples were incubated with standardized dilutions of NAGAB_HUMAN coming from a pool of 300 human sera from healthy subjects who did not show any sign of chronic inflammation or any disease. After 1 h incubation and exhaustive washing, complexes formed by NAGAB_HUMAN and purified DBP-MAF, used as positive control, or NAGAB_HUMAN and PMF, were revealed with horse radish peroxidase conjugate to rabbit antibody. The serum pool was mixed either with 200 ng of purified DBP-MAF, or with the fermented product in phosphate buffered saline (PBS) with the goal of studying the kinetics of interaction between NAGAB_HUMAN and DBP-MAF or NAGAB_HUMAN and the PMF. The compounds were incubated at 25C for 4, 24, 48, 72 and 120 h. Values for NAGAB_HUMAN binding activity in the absence of DBP-MAF or the PMF, with only PBS in the reaction mixture, were taken as 1.00. The experiment was repeated twice and the results reported in this study are the means of the two experiments. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.03.975524 doi: bioRxiv preprint intracellular domains are numbered IC1-IC4 , with the last domain pertaining to the intracellular carboxyl terminus. The degree of similarity in the aminoacid sequences is apparent in several regions of the proteins. Fig. 2, panel A, shows the infectivity determinant of HBV. The preS1 sequence in L protein (residues 2-48) is the sequence that specifically interacts with NTCP. Within this sequence, a highly conserved motif (9-NPLGF(F/L)P-15) is crucial for binding (11) . Fig. 2, panel B , shows the preS1 binding