A novel protease inhibitor of the alpha2-macroglobulin family expressed in the human epidermis

In the course of a large scale analysis of late-expressed genes in the human epidermis, we identified a new member of the alpha(2)-macroglobulin (alpha2M) protease inhibitor family, A2ML1 (for alpha(2)-macroglobulin-like 1). Like A2M and PZP, A2ML1 is located on chromosome 12p13.31. A2ML1 encodes a protein of 1454 amino acids, which fits the characteristics of alpha2Ms: 1) strong conservation in amino acid sequence including most of cysteine positions with alpha2M; 2) a putative central bait domain; 3) a typical thiol ester sequence. Northern blot and reverse transcriptase-PCR studies revealed a single 5-kb A2ML1 mRNA, mainly in the epidermis granular keratinocytes. A2ML1 is also transcribed in placenta, thymus, and testis. By Western blot analysis, alpha2ML1 is detected as a monomeric, approximately 180-kDa protein in human epidermis. In vitro keratinocyte differentiation is associated with increased expression levels. By immunohistochemistry, alpha2ML1 was detected within keratinosomes in the granular layer of the epidermis, and as a secreted product in the extracellular space between the uppermost granular layer and the cornified layer. Recombinant alpha2ML1 displayed inhibitory activity toward chymotrypsin, papain, thermolysin, subtilisin A, and to a lesser extent, elastase but not trypsin. Incubation with chymotrypsin and the chymotrypsin-like kallikrein 7 protease indicated that alpha2ML1 binds covalently to these proteases, a feature shared with other members of the family. Therefore, alpha2ML1 is the first alpha2M family member detected in the epidermis. It may play an important role during desquamation by inhibiting extracellular proteases.