Full length article Yellow head virus binding to cell surface proteins from Penaeus monodon hemocytes Phattara-orn Havanapan a , Suparat Taengchaiyaphum a , Apichai Bourchookarn b , Albert J. Ketterman a , Chartchai Krittanai a, * a Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom, Thailand b Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla University, Pattani, Thailand article info Article history: Received 31 March 2014 Received in revised form 4 August 2014 Accepted 8 August 2014 Available online 27 August 2014 Keywords: Penaeus monodon Yellow head virus Biotinylation Hemocyte Crustin abstract Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using BiotineNSHeLC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect bio- tinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. CrustinPm1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustinPm1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. CrustinPm1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Hemocytes, comparable to blood cells in mammals, are the primary target of cellular immune response in crustaceans. There are two classes of hemocytes based on granularity, agranular (hy- aline) and granule-containing hemocytes (semigranular and gran- ular cells). To date, our understanding of the molecular response of the shrimp-immune system via each specific cell type of hemocyte is still unclear. The roles of each hemocyte subtype in immune response are controversial and dependent on infecting agents of the crustaceans [1,2]. In Penaeus merguiensis, granular hemocytes were observed to be the target of white spot syndrome virus (WSSV) [3]. In our group, we found that yellow head virus (YHV) specifically interacts with granular cells at 1 h post infection (hpi), which is the early phase of infection. At 12 hpi, semigranular he- mocytes showed higher levels of YHV infection. However, no YHV was detected in hyaline hemocytes similar to what was observed in WSSV [3]. Granule-containing hemocytes appeared to be specif- ically targeted for yellow head virus entry. Therefore, we postulated that the process of viral entry into permissive cells, such as granular hemocytes, is mediated by cell specific binding proteins. Recently, several methods have been reported for selective extraction of membrane proteins involving subcellular fraction- ation using chemical [4] and membrane protein labeling (e.g. CyDye DIGE Fluor and biotin). The elucidation of membrane pro- teins has been carried out by a cell surface biotinylation method both in vertebrates and invertebrates [5e7]. Cell surface bio- tinylation has emerged as an important tool for studying the expression and regulation of receptors, transporters, cell differen- tiation and distribution of membrane proteins in epithelial cells [8,9]. In the present report, the differentially expressed membrane proteins of granule-containing hemocytes and hyaline hemocytes were comparatively investigated using biotinylation and gel-based proteomics followed with identification by mass spectrometry. These identified membrane and membrane-associated proteins provide information not only concerning basic roles in fundamental biological processes, but also highlight possible novel targeted proteins for viral entry into permissive hemocytes. This research * Corresponding author. Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom 73170, Thailand. Tel.: þ66 2 4419003x1410; fax: þ66 2 441 1013. E-mail address: chartchai.kri@mahidol.ac.th (C. Krittanai). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi http://dx.doi.org/10.1016/j.fsi.2014.08.014 1050-4648/© 2014 Elsevier Ltd. All rights reserved. Fish & Shellfish Immunology 41 (2014) 126e136