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Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.

Wu Z, Hansmann B, Meyer-Hoffert U, Gläser R, Schröder JM - PLoS ONE (2009)

Bottom Line: We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta.We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis.Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany.

ABSTRACT
Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca(2+) stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors.

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Western blot analysis of filaggrin-2 in human skin.(A) Total proteins were individually extracted from SC and abdominal epidermis, separated on an SDS-10% PAGE and blotted on a nitrocellulose membrane. The FLG2 protein was detected by using purified goat anti-FLG2 polyclonal antibody and HRP-conjugated mouse anti-goat IgG as the second antibody. Proteins were extracted sequentially from abdominal epidermis with PBS buffer (lane 1), SDS buffer (62.5 mM Tris, 10% glycerol, 5% SDS) (lane 2), and SDS/DTT buffer (SDS buffer plus 10 mM DTT) (lane 3), and from trypsin-digested skin sample (lane 4); alternatively, proteins were sequentially extracted from SC with PBS buffer (lane 5), SDS buffer (lane 6) and SDS/DTT buffer (lane 7), and from trypsin-digested SC sample (lane 8). In all other lanes, 5 µl of protein extracts were loaded. The approximate size of the protein was determined using high-molecular-weight standards. The experiment was performed twice and a representative example is shown. (B) As a control, Western blot analysis of the purified recombinant FLG2 with a theoretical monomolecular mass of 15.66 kDa was performed under the same assay conditions. Different amounts of protein were loaded as indicated. The ladder-like bands appeared with higher amounts of protein.
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pone-0005227-g007: Western blot analysis of filaggrin-2 in human skin.(A) Total proteins were individually extracted from SC and abdominal epidermis, separated on an SDS-10% PAGE and blotted on a nitrocellulose membrane. The FLG2 protein was detected by using purified goat anti-FLG2 polyclonal antibody and HRP-conjugated mouse anti-goat IgG as the second antibody. Proteins were extracted sequentially from abdominal epidermis with PBS buffer (lane 1), SDS buffer (62.5 mM Tris, 10% glycerol, 5% SDS) (lane 2), and SDS/DTT buffer (SDS buffer plus 10 mM DTT) (lane 3), and from trypsin-digested skin sample (lane 4); alternatively, proteins were sequentially extracted from SC with PBS buffer (lane 5), SDS buffer (lane 6) and SDS/DTT buffer (lane 7), and from trypsin-digested SC sample (lane 8). In all other lanes, 5 µl of protein extracts were loaded. The approximate size of the protein was determined using high-molecular-weight standards. The experiment was performed twice and a representative example is shown. (B) As a control, Western blot analysis of the purified recombinant FLG2 with a theoretical monomolecular mass of 15.66 kDa was performed under the same assay conditions. Different amounts of protein were loaded as indicated. The ladder-like bands appeared with higher amounts of protein.

Mentions: To get a more deep insight into its biological function, we performed Western blot analyses to examine FLG2 expression and protein processing in the skin (Fig. 7A). Using detergent-free conditions, we observed very faint staining in both the epidermal extract (∼65 kDa) and the SC extract (∼65 kDa and ∼15 kDa) (lane 1 and 5). When extraction was performed in the presence of SDS, the epidermal extract exhibited two strong bands, one corresponding to the expected molecular mass of full-length FLG2 (∼248 kDa) and another corresponding to a fragment at ∼65 kDa (lane 2). Subsequent treatment of the extract with DTT-containing buffer resulted in extraction of additional FLG2 bands corresponding to 40, 30, 25, and 20 kDa size, respectively (lane 3). In contrast, SC extracts exhibited different band patterns with stronger small-size FLG2 bands, especially a 15-kDa size band, but without a clear band at the size of full length FLG2 (lane 6 and 7). Interestingly, extracts obtained from further treatment of the SC sediment with trypsin did not abolish any FLG2-immunostaining but resulted in broad intense bands between 45 and 72 kDa (lane 8).


Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.

Wu Z, Hansmann B, Meyer-Hoffert U, Gläser R, Schröder JM - PLoS ONE (2009)

Western blot analysis of filaggrin-2 in human skin.(A) Total proteins were individually extracted from SC and abdominal epidermis, separated on an SDS-10% PAGE and blotted on a nitrocellulose membrane. The FLG2 protein was detected by using purified goat anti-FLG2 polyclonal antibody and HRP-conjugated mouse anti-goat IgG as the second antibody. Proteins were extracted sequentially from abdominal epidermis with PBS buffer (lane 1), SDS buffer (62.5 mM Tris, 10% glycerol, 5% SDS) (lane 2), and SDS/DTT buffer (SDS buffer plus 10 mM DTT) (lane 3), and from trypsin-digested skin sample (lane 4); alternatively, proteins were sequentially extracted from SC with PBS buffer (lane 5), SDS buffer (lane 6) and SDS/DTT buffer (lane 7), and from trypsin-digested SC sample (lane 8). In all other lanes, 5 µl of protein extracts were loaded. The approximate size of the protein was determined using high-molecular-weight standards. The experiment was performed twice and a representative example is shown. (B) As a control, Western blot analysis of the purified recombinant FLG2 with a theoretical monomolecular mass of 15.66 kDa was performed under the same assay conditions. Different amounts of protein were loaded as indicated. The ladder-like bands appeared with higher amounts of protein.
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Related In: Results  -  Collection

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pone-0005227-g007: Western blot analysis of filaggrin-2 in human skin.(A) Total proteins were individually extracted from SC and abdominal epidermis, separated on an SDS-10% PAGE and blotted on a nitrocellulose membrane. The FLG2 protein was detected by using purified goat anti-FLG2 polyclonal antibody and HRP-conjugated mouse anti-goat IgG as the second antibody. Proteins were extracted sequentially from abdominal epidermis with PBS buffer (lane 1), SDS buffer (62.5 mM Tris, 10% glycerol, 5% SDS) (lane 2), and SDS/DTT buffer (SDS buffer plus 10 mM DTT) (lane 3), and from trypsin-digested skin sample (lane 4); alternatively, proteins were sequentially extracted from SC with PBS buffer (lane 5), SDS buffer (lane 6) and SDS/DTT buffer (lane 7), and from trypsin-digested SC sample (lane 8). In all other lanes, 5 µl of protein extracts were loaded. The approximate size of the protein was determined using high-molecular-weight standards. The experiment was performed twice and a representative example is shown. (B) As a control, Western blot analysis of the purified recombinant FLG2 with a theoretical monomolecular mass of 15.66 kDa was performed under the same assay conditions. Different amounts of protein were loaded as indicated. The ladder-like bands appeared with higher amounts of protein.
Mentions: To get a more deep insight into its biological function, we performed Western blot analyses to examine FLG2 expression and protein processing in the skin (Fig. 7A). Using detergent-free conditions, we observed very faint staining in both the epidermal extract (∼65 kDa) and the SC extract (∼65 kDa and ∼15 kDa) (lane 1 and 5). When extraction was performed in the presence of SDS, the epidermal extract exhibited two strong bands, one corresponding to the expected molecular mass of full-length FLG2 (∼248 kDa) and another corresponding to a fragment at ∼65 kDa (lane 2). Subsequent treatment of the extract with DTT-containing buffer resulted in extraction of additional FLG2 bands corresponding to 40, 30, 25, and 20 kDa size, respectively (lane 3). In contrast, SC extracts exhibited different band patterns with stronger small-size FLG2 bands, especially a 15-kDa size band, but without a clear band at the size of full length FLG2 (lane 6 and 7). Interestingly, extracts obtained from further treatment of the SC sediment with trypsin did not abolish any FLG2-immunostaining but resulted in broad intense bands between 45 and 72 kDa (lane 8).

Bottom Line: We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta.We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis.Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany.

ABSTRACT
Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca(2+) stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors.

Show MeSH
Related in: MedlinePlus