A Novel Protease Inhibitor of the α2-Macroglobulin Family Expressed in the Human Epidermis

Abstract

In the course of a large scale analysis of late-expressed genes in the human epidermis, we identified a new member of the ␣ 2 -macroglobulin (␣2M) protease inhibitor family, A2ML1 (for ␣ 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 ␣2Ms: 1) strong conservation in amino acid sequence including most of cysteine positions with ␣2M; 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, ␣2ML1 is detected as a monomeric, ϳ180-kDa protein in human epidermis. In vitro keratinocyte differentiation is associated with increased expression levels. By immunohistochemistry, ␣2ML1 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 ␣2ML1 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 ␣2ML1 binds covalently to these proteases, a feature shared with other members of the family. Therefore, ␣2ML1 is the first ␣2M family member detected in the epidermis. It may play an important role during desquamation by inhibiting extracellular proteases.Regulation of proteolytic enzyme activity is essential for cell and tissue homeostasis. In epidermis, proteolysis of adhesive structures is a prerequisite for desquamation. Several members of the four protease classes with suggested roles in desquamation have been described in epidermis (reviewed in Ref. 1). A wide variety of protease inhibitors is also present in the intercellular spaces of the stratum corneum and participates in the regulation of desquamation-associated proteolysis. Disturbance of the protease-antiprotease balance may have dramatic consequences as demonstrated by the discovery of the serine protease inhibitor Kazal type 5 (SPINK5) also known as lymphoepithelial Kazal type-related inhibitor (LEKTI) as the defective gene in Netherton syndrome (2). The importance of regulated proteolysis in epidermis has also been reported in mouse models. Targeted epidermal overexpression of the serine protease kallikrein 7 (KLK7), 2 also known as stratum corneum chymotryptic enzyme (SCCE), results in a severe phenotype (3). Conversely, a null mutation in the mouse cystatin M/E gene (Cst6), encoding a cysteine protease inhibitor, induces neonatal lethality and abnormalities in cornification and desquamation, highlighting the essential role for protease inhibitors during the final stages of epidermal differentiation (4).In the course of a large scale search for genes specifically expressed by the last transcriptionally activ...

Identification of Differentially Expressed Proteins in the Cervical Mucosa of HIV-1-Resistant Sex Workers

“…44 Serpin B1 and A2ML1 have specific elastase inhibitory properties. 36,45 Although not identified in this study, human neutrophil elastase (HNE), a serine protease, is known to be present in the cervicovaginal compartment. 46 It has been suggested that it can increase the risk of HIV-1 infection by enhancing myeloid-related protein (MRP-8) expression, 47 an inflammatory protein in cervicovaginal fluid known to stimulate HIV-1 production.…”

Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome

“…These proteins display a unique trap mechanism of inhibition, by which the A2M inhibitor undergoes a major conformational change upon its cleavage by a protease, thereby trapping the protease and blocking it from subsequent substrate binding. 19 Moreover, A2ML1 binds to the lipoprotein receptor-related protein 1 (LRP1) receptor, 20 an upstream activator of the MAPK/ ERK cascade. 21 Additionally, LRP1 directly interacts with CBL.…”

“…Mutations identified were checked for de novo occurrence whenever parental DNAs were available. For the 140 individuals with NS or a clinically related phenotype selected from the ISS and UCSC, Rome, Italy, exons 15,16,19,24,25,26,29, 31 and 32 and flanking intronic sequences were considered, based on the identification of possible pathogenic mutations in the first cohort. For all variants detected, an in silico-based method was used to assess the effect of the mutation (Alamut software, version 2.1; http://www.interactivebiosoftware.com/) in addition to an assessment of variant pathogenicity according to guidelines by the CMGS and VKGL, for the British and Dutch Molecular Genetic Societies, respectively.…”

Lamellar Bodies of Human Epidermis

“…␤-glucocerebrosidase also known as glucosylceramidase, acid sphingomyelinase, and secretory phospho-lipase A 2 ) that make the upper layers waterproof (8,9), structural proteins like corneodesmosin that optimizes corneocyte cohesion (10), proteases (kallikreins and cathepsins), glycosidases and protease inhibitors (e.g. elafin, secretory leucocyte protease inhibitor, lympho-epithelial kazal-type-related inhibitor, and ␣ 2 -macroglobulin-like) involved in the control of desquamation (11,12), and antimicrobial peptides (defensins and cathelicidin) (13,14), at the stratum granulosum/ stratum corneum interface. The secreted proteins seem to be delivered by independent trafficking of various cargoes (4).…”