همایش ملی بیوانفورماتیک ایران

صفحه اصلی / 4th international edition and 13th Iranian Conference on Bioinformatics

Computational Screening of Signal Peptides for Optimized Secretory Production of Recombinant Lutheran b (Lub) Blood Group Antigen in Human Cell Lines

نویسندگان :

Kosar Asadi¹ Maryam Latifi² Mansoureh Shahbazi Dastjerdeh³

1- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran 2- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran 3- Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

کلمات کلیدی :

Protein Sorting Signals،Computational Biology،Recombinant Proteins،Blood Group Antigen

چکیده :

Background: In current blood transfusion systems, the detection of antibodies against red blood cell antigens relies on panels of red blood cells that have been phenotyped for most blood group antigens. Due to the high diversity of antigens on the surface of red blood cells, identifying antibody specificity depends on the lack of reactivity of the antibody with antigen-negative cells. Consequently, detecting alloantibodies against high-prevalence antigens is challenging in immunohematology laboratories with conventional serological methods due to the unavailability of negative cells. Recombinant blood group proteins, through their specific interaction with antibodies in the serum and their ability to inhibit agglutination between the target antibody and red blood cells in the panel, enable precise and rapid identification of alloantibody specificity without the need for additional time-consuming tests. This approach reduces the referrals to reference laboratories and facilitates faster and safer blood provision for patients (Flegel and Srivastava 2021). Lutheran B (Lub) is a type I integral membrane glycoprotein and a high-prevalence blood group antigen, present in more than 99% of the global population. Studies have demonstrated that the soluble recombinant form of Lub consisting of its extracellular domains can specifically bind to alloantibodies directed against it. This property makes recombinant Lub a valuable tool in immunohematology for resolving complex antibody identification cases particularly when Lub negative red blood cells are not available (Ridgwell, Dixey et al. 2007, Seltsam, Grüger et al. 2007). Human cell lines such as HEK293, are well-suited expression hosts for producing recombinant blood group proteins (rBGPs), including Lub, as they provide the necessary cellular machinery to perform post-translational modifications and proper folding, closely resembling native human blood group antigens (Seltsam and Blasczyk 2016). Efficient secretion of recombinant proteins in these cell lines mainly depends on application of proper signal peptide (SP). Typically, SPs range between 15 and 30 residues, carrying information for the protein secretion, hence, directing the molecule into the lumen of the ER (Pool 2022). To our knowledge, there is still a lack of understanding about the best SP for the secretion of recombinant Lub in mammalian cell lines. Here, we focused on computational selection of the best SP for Lub among the 74 candidate signal peptides. Method: The amino acid sequence of Lub was retrieved from the Uniprot server. Furthermore, 74 eukaryote SPs were collected from previous studies. The presence and location of the cleavage site were predicted for each SP fused to N-terminal of Lub using SignalP 6.0. The physicochemical features including GRAVY, Aliphatic index, Instability index, and Net positive charge were predicted by the ProtParam server. Solubility of each SP was predicted using DeepLoc 2.1 server which uses a deep learning method for the prediction of subcellular localization of eukaryotic proteins. Results and discussion: Computational analysis of SPs regarding cleavability, stability, and physicochemical features affecting protein secretion, resulted in selection of trypsinogen-2, CALR, and native G, respectively, as the best theoretical candidate signal peptides with the highest GRAVY for production of recombinant Lub blood group antigen in mammalian cell lines.