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SPINK9: a selective, skin-specific Kazal-type serine protease inhibitor.
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.ORCID iD: 0000-0001-7175-1336
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Dermatology and Venerology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
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2009 (English)In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 129, no 7, 1656-1665 p.Article in journal (Refereed) Published
Abstract [en]

A previously unreported Kazal-type serine protease inhibitor, serine protease inhibitor Kazal type 9 (SPINK9), was identified in human skin. SPINK9 expression was strong in palmar epidermis, but not detectable or very low in non palmoplantar skin. Analysis of a human cDNA panel showed intermediate expression in thymus, pancreas, liver, and brain, and low or undetectable expression in other tissues. Using kallikrein-related peptidases (KLKs) 5, 7, 8, and 14, thrombin, trypsin, and chymotrypsin, inhibition with recombinant SPINK9 was seen only for KLK5 using low molecular weight substrates, with an apparent K(i) of 65 nM. Also KLK5 degradation of fibrinogen was totally inhibited by SPINK9. Slight inhibition of KLK8 using fibrinogen substrate could be observed using high concentrations of SPINK9. Analyses by surface plasmon resonance showed heterogeneous binding to SPINK9 of KLK5 and KLK8, but no binding of KLK7 or KLK14. KLK5 has been suggested to play a central role in skin desquamation as an initiating activating enzyme in proteolytic cascades formed by KLKs. An apparently KLK5-specific inhibitor, such as SPINK9, may play a significant regulatory role in such cascades. We suggest a possible role for SPINK9 in the site-specific epidermal differentiation of palms and soles.

Place, publisher, year, edition, pages
Nature Publishing Group , 2009. Vol. 129, no 7, 1656-1665 p.
National Category
Dermatology and Venereal Diseases
URN: urn:nbn:se:umu:diva-35834DOI: 10.1038/jid.2008.448PubMedID: 19194479OAI: diva2:349579
Available from: 2010-09-07 Created: 2010-09-07 Last updated: 2016-08-22Bibliographically approved
In thesis
1. Kallikrein-related peptidases in human epidermis: studies on activity, regulation, and function
Open this publication in new window or tab >>Kallikrein-related peptidases in human epidermis: studies on activity, regulation, and function
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction. The outermost layer of the epidermis, the stratum corneum (SC), plays a fundamental role in our defense against microorganisms, chemicals, and dehydration. The SC is composed of tightly packed keratinized skin cells, corneocytes. For a functioning skin it is essential that corneocytes are constantly shed (desquamated). Kallikrein-related peptidase (KLK) 5 and KLK7 may be important in the desquamation process through degradation of desmosomal proteins. Severe hereditary diseases, where inhibition of KLK5 and/or KLK7 is missing, points to the importance of regulation of protease activity. KLKs may be regulated in various ways: tissue expression, activation of proforms, specific inhibitors, and physico-chemical properties like pH. Besides their involvement in desquamation, KLKs may also be important in immune defense and inflammation by processing of mediators and via activation of proteinase-activated receptors (PARs).

Aims. 1. To identify and characterize previously unknown proteases in the SC. 2. To further characterize KLK5 and KLK7 with special focus on activation mechanisms. 3. To identify new inhibitors of KLKs in human SC. 4. To further characterize KLKs regarding effects of various inhibitors and substrates. 5. To study possible functions of KLKs in inflammation, in particular via activation of PAR-2.

Methods. Plantar SC was used as a source for purification of proteins. Recombinant proteins were produced in different expression systems (insect cells, yeast cells, and bacteria). Different activity assays and kinetic studies were performed. Tissue expression was studied by immunohistochemistry, immunoblot and PCR. PAR-2 activation was studied by measurement of intracellular [Ca2+] and immunofluorescense in KNRK-PAR2 cells.

Results. Active KLK14 was purified from extracts of plantar SC. KLK14 showed a superior catalytic efficiency as compared to KLK5 when measuring trypsin-like activity. This indicated that KLK14, despite being present in low amounts in skin, may have great relevance for skin physiology. Among enzymes tested only KLK5 showed autocatalytic activity and is so far the only enzyme found in SC that can activate proKLK7. KLK5 could also activate proKLK14. This together with studies of pH dependence on activation placed KLK5 as a possible key activating enzyme in a proposed proteolytic cascade in the SC. In plantar SC extracts we have also identified the novel Kazal-type serine protease inhibitor 9 (SPINK9). Our results indicate that SPINK9 is preferentially expressed in palmo-plantar skin and specific for KLK5. Differences found regarding substrate specificity and inhibition profile can be useful in evaluating the contribution of individual KLKs to the proteolytic activity in crude SC extracts. One interesting finding was that KLK8, present at high protein levels in the epidermis, could not be inhibited by any protease inhibitor found in the extracts. PAR-2 activation studies showed that KLK5 and 14 but neither KLK7 nor 8 can activate PAR-2. Immunohistochemistry preferentially detected KLK14 in intraepidermal parts of the sweat ducts and in dermal sweat glands but we could also show coexpression of KLK14 and PAR-2 in the SC and stratum granulosum of the epidermis in inflammatory skin disorders. To summarize, KLK involvement in desquamation may be dependent on a proteolytic activation cascade regulated by an intrinsic pH gradient and specific inhibitors present in SC. Another possible function of KLKs is as mediators of inflammation through activation of PAR-2.

Place, publisher, year, edition, pages
Umeå: Dermatologi och venereologi, 2008. 80 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1174
desquamation, epidermis, stratum corneum, kallikrein-related peptidase, KLK, serine protease inhibitors
National Category
Dermatology and Venereal Diseases
urn:nbn:se:umu:diva-1644 (URN)978-91-7264-555-4 (ISBN)
Public defence
2008-05-29, Hörsal Betula, Byggnad 6M, Umeå universitet, SE-901 85 Umeå, 09:00 (English)
Available from: 2008-05-12 Created: 2008-05-12 Last updated: 2011-01-25Bibliographically approved

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Brattsand, MariaStefansson, KristinaHubiche, ThomasNilsson, Stefan KEgelrud, Torbjörn
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