Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1
Abstract Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initi...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2023-12-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-48856-1 |
| _version_ | 1797398044548792320 |
|---|---|
| author | Marta Surbek Tessa Van de Steene Attila Placido Sachslehner Bahar Golabi Johannes Griss Sven Eyckerman Kris Gevaert Leopold Eckhart |
| author_facet | Marta Surbek Tessa Van de Steene Attila Placido Sachslehner Bahar Golabi Johannes Griss Sven Eyckerman Kris Gevaert Leopold Eckhart |
| author_sort | Marta Surbek |
| collection | DOAJ |
| description | Abstract Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1. |
| first_indexed | 2024-03-09T01:19:02Z |
| format | Article |
| id | doaj.art-e17a93f2ae7644e1888d6ca051394575 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T01:19:02Z |
| publishDate | 2023-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e17a93f2ae7644e1888d6ca0513945752023-12-10T12:17:44ZengNature PortfolioScientific Reports2045-23222023-12-0113111510.1038/s41598-023-48856-1Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1Marta Surbek0Tessa Van de Steene1Attila Placido Sachslehner2Bahar Golabi3Johannes Griss4Sven Eyckerman5Kris Gevaert6Leopold Eckhart7Department of Dermatology, Medical University of ViennaVIB Center for Medical Biotechnology Center, VIB, Ghent UniversityDepartment of Dermatology, Medical University of ViennaDepartment of Dermatology, Medical University of ViennaDepartment of Dermatology, Medical University of ViennaVIB Center for Medical Biotechnology Center, VIB, Ghent UniversityVIB Center for Medical Biotechnology Center, VIB, Ghent UniversityDepartment of Dermatology, Medical University of ViennaAbstract Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1.https://doi.org/10.1038/s41598-023-48856-1 |
| spellingShingle | Marta Surbek Tessa Van de Steene Attila Placido Sachslehner Bahar Golabi Johannes Griss Sven Eyckerman Kris Gevaert Leopold Eckhart Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 Scientific Reports |
| title | Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 |
| title_full | Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 |
| title_fullStr | Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 |
| title_full_unstemmed | Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 |
| title_short | Cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase-1 |
| title_sort | cornification of keratinocytes is associated with differential changes in the catalytic activity and the immunoreactivity of transglutaminase 1 |
| url | https://doi.org/10.1038/s41598-023-48856-1 |
| work_keys_str_mv | AT martasurbek cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT tessavandesteene cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT attilaplacidosachslehner cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT bahargolabi cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT johannesgriss cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT sveneyckerman cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT krisgevaert cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 AT leopoldeckhart cornificationofkeratinocytesisassociatedwithdifferentialchangesinthecatalyticactivityandtheimmunoreactivityoftransglutaminase1 |