A novel peptide 'T14' reflects age and photo-aging in human skin

Abstract

T14 is a 14mer peptide derived from the C-terminus of acetylcholinesterase (AChE). Once cleaved, it is independently bioactive of the parent molecule and enhances calcium influx in different cell types, in a range of scenarios: it binds to an allosteric site selectively on the alpha-7 receptor, where it modulates calcium influx and is thus a potential trophic agent, as already reported in a range of normal developmental scenarios. However, if inappropriately activated, this erstwhile beneficial effect converts to a toxic one, resulting in pathologies as disparate as Alzheimer's and various metastatic cancers. Given that epidermal keratinocyte cells have the same ectodermal origin as brain cells, as well as expressing AChE and the alpha-7 receptor, we have explored whether T14 plays a comparable role. Here we report that the T14 immunoreactivity is detectable in human keratinocytes with levels inversely related to age: this decrease is even more apparent with chronic photo-exposure and thus accelerated skin aging. We conclude that T14, an agent promoting cell growth and renewal in other parts of the body, also operates in skin, Moreover, monitoring of keratinocyte T14 levels might offer further insights into the now well reported link between degenerative diseases and epidermal cell profile.

Keywords: acetylcholinesterase peptide; age; keratinocyte; photo-aging; skin.

Figure 1

Specificity of T14 antibody. (A) Comparison of T14 antibody specificity using ELISA to detect peptides of various lengths with the C-terminus capped with W or K. (B) List of peptides used with amino acids sequences (epitope of the antibody -VHWK highlighted in red). (C) Dose-response of different peptides at the nanomolar range to determine the importance of the length and the epitope. (D) Dose response of T14, T30, T15 and AChE at nanomolar range. (E) Effect of trypsin on full AChE, cleaving T14 now detectable by the antibody.

Figure 2

Detection of T14 in skin. (A) Immunohistochemistry of AChE T14 peptide expression is observed in both the dermis and epidermis. In the epidermis the expression is high in human skin of young subjects (16 to 31 years old), decreases in skin of middle-aged subjects (37 to 45 years old) and further declines in skin of aged subjects (50 to 70 years old). (B) Average number of epidermal cells with a positive AChE T14 peptide antibody stain. Young skin samples have a significantly higher expression (p value of 0.00018) of T14 positive cells compared to its expression in both middle-aged and aged skin samples. (C) Average number of epidermal cells with a positive AChE T14 peptide antibody stain normalized by epidermal area. Young skin samples have a significantly higher expression (p value of 0.0000015) of T14 positive cells per epidermal area compared to its expression in both middle-aged and aged skin samples. (D) Peptide block of T14 and anti-T14 staining of young PP skin sample and aged PP skin sample. Peptide successfully blocked T14 binding of epitope for both young and aged photo-protected skin tissues. N=10 in each group.

Figure 3

Effect of UV-light on levels of T14 in skin. (A) Immunohistochemistry of AChE T14 peptide expression is higher in aged photo-protected (50 to 70 years old) skin compared to photo-aged skin (50 to 70 years old. N=10 in each group. (B) Average number of epidermal cells with a positive AChE T14 peptide antibody stain. Aged (photo-protected) skin samples have a significantly higher expression (p value of 0.00265) of T14 positive cells compared to its expression in age matched, photo-aged skin samples. (C) Average number of epidermal cells with a positive AChE T14 peptide antibody stain normalized by epidermal area. Aged (photo-protected) skin samples have a significantly higher expression (p value of 0.00176) of T14 positive cells per epidermal area compared to its expression in age-matched photo-exposed skin. (D) Immunohistochemistry of AChE T14 peptide expression is higher in 1 outlier of the aged group.