Quantification of Eight Tissue Kallikreins in the Stratum Corneum and Sweat

Komatsu, Naoya; Tsai, Brian; Sidiropoulos, Michael; Saijoh, Kiyofumi; Levesque, Michael A.; Takehara, Kazuhiko; Diamandis, Eleftherios P.

doi:10.1038/sj.jid.5700146

Cited by 47 publications

(60 citation statements)

References 17 publications

(16 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our findings were in good agreement with the previously reported KLK levels measured by ELISA. For example, concentration of endogenous KLK6 in sweat previously measured with ELISA was found in the range 1-50 g/g of total protein (48). To the best of our knowledge, the present study reports for the first time the mass spectrometric detection of KLKs 1, 6, 9, 10, and 13 in human sweat.…”

Section: Discussionmentioning

confidence: 72%

“…Therefore, normalization of results for total protein is crucial. Kallikreins 5,6,7,8,10,11,13, and 14 have been previously quantified in sweat by ELISA (48) and KLKs 5,7,8, and 11 have been previously detected by shotgun proteomics (49 -51). Using the proposed SRM assay, we were able to detect and quantify nine KLKs in sweat (KLKs 1, 5, 6, 7, 8, 9, 10, 11, and 13).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

Karakosta

Soosaipillai

Diamandis

et al. 2016

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs. Molecular & Cellular

show abstract

Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

Karakosta

Soosaipillai

Diamandis

et al. 2016

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…Nonetheless, KLK5 activation of pro-KLK8 may be important physiologically as normal human epidermal cell turnover occurs in the span of 2-4 weeks. Furthermore, expression data suggest that immunoreactive KLK8 concentration in normal human SC tissue extracts is about 4-fold higher than KLK5 (21). Hence, this activation process may occur quicker had we used a pro-KLK8:KLK5 molar ratio of 1:4 or 1:1 instead of 1:10.…”

Section: Mat-klk8 Autodegradation Leads To In Vitro Enzymaticmentioning

confidence: 99%

“…We recently showed that additional trypsin-like KLK peptidases, including KLK6, KLK8, KLK10, KLK11, and KLK13, co-exist with KLK5, KLK7, and KLK14 in stratum corneum tissues, hair follicles, and sebaceous and sweat glands (20,21). Interestingly, we found KLK8 to be the most abundant trypsin-like KLK in normal stratum corneum and sweat, among the seven KLKs tested (21).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

Eissa

Amodeo

Smith

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg 164 . Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1-P2. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.

show abstract

The Stratum Corneum: The Rampart of the Mammalian Body

Nishifuji

Yoon

2013

Advances in Veterinary Dermatology

View full text Add to dashboard Cite

Quantification of Eight Tissue Kallikreins in the Stratum Corneum and Sweat

Cited by 47 publications

References 17 publications

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

The Stratum Corneum: The Rampart of the Mammalian Body

Contact Info

Product

Resources

About