Localization of Hair Shaft Protein VSIG8 in the Hair Follicle, Nail Unit, and Oral Cavity

TO THE EDITOR The molecular bases of diseases of the epidermal appendages are gradually being elucidated as genes encoding their constituents and regulation are being defined. A direct correspondence between altered protein sequence and aberrant hair shaft structure is evident in cases such as monilethrix (Schweizer, 2006). In others, gene defects lead to altered development and thus perturbed regulation of components. Identification of prominent constituents will speed recognition of genes whose defects contribute directly to aberrant structure or indirectly by exacerbating the effects of deficiencies in other genes. The present work helps characterize the component V-set and immunoglobulin domain containing 8 (VSIG8) in hair shaft and nail plate to assist in understanding its possible relation to disease states. Protein components of hair shaft and nail plate were known for many years to consist largely of keratin intermediate filaments and keratin-associated proteins. Other components, especially those subject to transglutaminase cross-linking, were difficult to identify. Using mass spectrometry-based shotgun proteomics, isolation of proteins is no longer necessary for their identification. Indeed, aggregates of dozens, even hundreds of proteins, are amenable to analysis. Such analysis confirmed that the Woolly hair syndrome in one family is not due to a defective structural protein component, but rather is a consequence of lipase H mutations (Shimomura et al., 2009). Similarly, mouse strains can be distinguished by proteomic analysis of their pelage hair (Rice et al., 2009). Our initial proteomic analysis of human hair identified a prominent protein component for which little information was available, VSIG8 (Lee et al., 2006). A specific antipeptide rabbit antiserum was raised (Antibodies, Davis, CA) to a highly conserved unique peptide segment near the amino-terminus of the predicted protein sequence (CSAVRINGDGQEVLYLAEGDNVRL, residues 20–42 with an additional amino-terminal C permitting attachment to the KLH carrier protein) of VSIG8. A complementary DNA clone of the predicted coding sequence (NM_001013661.1) was prepared commercially (Origene Technologies, Rockville, MD), inserted into a mammalian expression vector, and used in transient transfections of HeLa and human embryonic kidney 293 cells as a positive control. Immunoblotting revealed a strongly immunoreactive band with mobility corresponding to E45 kDa, the predicted mass. Minor immunoreactive bands with mobilities similar to keratins were not observed when the antiserum was absorbed with protein solubilized from hair shaft and skin callus with SDS and dithioerythritol. As VSIG8 protein is not solubilized in this way (Rice et al, 2010), absorption did not impair the use of antiserum in this study. The coding region was subjected to sitedirected mutagenesis (QuikChange kit, Stratagene, La Jolla, CA) converting either L at position 32 to M (mouse sequence) or Y at position 33 to F (rat sequence). Extracts of cultures transfected with all the constructs contained similarly immunoreactive bands. To optimize the immunohistochemical (IHC) analysis, we tested mouse skin biopsy specimens preserved in five fixatives. Fekete’s solution (100 ml of 70% alcohol, 10 ml formalin, and 5 ml glacial acetic acid) worked well, enabling us to use archival samples. For tissue distribution analysis, we recut blocks encompassing all major and minor organs from one male NON/ ShiLtJ mouse at 12 months of age. VSIG8 protein expression was observed in the hair follicle at specific stages of the hair cycle, the oral mucosa, and the nail matrix. Expression in the hair follicle and shaft was found in the cuticle and cortex layers of the hair (Figure 1a), where the labeling appeared intracellular and at the cell periphery (Figure 1b). IHC analysis revealed that VSIG8 was located in the projections of the cortex between medullary cells (Figure 1c and d), a poorly understood structure that is deficient in mice homozygous for the hair interior defect due to a mutation in Soat1 (Wu et al., 2010). To determine whether VSIG8 was expressed at all stages of the hair cycle or only in specific stages and anatomical sites, synchronized (wax-stripped) hair follicles (Sundberg and Silva, in press) were tested. Reactivity was restricted as we initially found in the hair follicle and hair shaft, and positive signals were limited to the late anagen and early catagen stages of the hair cycle (Figure 1e–k). The nail unit is a highly specialized structure with a variety of functions (Fleckman, 2005). Although numerous genes involved in normal hair follicle and shaft development are also involved in the nail (Kitahara and Ogawa, 1997; De Berker et al., 2000), much remains unknown. We found that VSIG8 was not limited to the hair shaft and precortex of the hair follicle bulb, but was also found in the superficial layers of the nail matrix (Figure 1l–m). Messenger RNA encoding VSIG8 protein was detected previously at LETTERS TO THE EDITOR