Activation Profiles and Regulatory Cascades of the Human Kallikrein-related Peptidases

Hyesook Yoon,Gurunathan Laxmikanthan,Jihun Lee,Sachiko I Blaber,Aaron Rodriguez,Joshua M Kogot,Isobel A Scarisbrick,Michael Blaber

doi:10.1074/jbc.m705190200

Hyesook Yoon, Gurunathan Laxmikanthan + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m705190200

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Abstract

The human kallikrein (KLK)-related peptidases are the largest family of serine peptidases, comprising 15 members (KLK1-15) and with the majority (KLK4-15) being identified only within the last decade. Members of this family are associated with important diseased states (including cancer, inflammation, and neurodegeneration) and have been utilized or proposed as clinically important biomarkers or therapeutic targets of interest. All human KLKs are synthesized as prepro-forms that are proteolytically processed to secreted pro-forms via the removal of an amino-terminal secretion signal peptide. The secreted inactive pro-KLKs are then activated extracellularly to mature peptidases by specific proteolytic release of their amino-terminal propeptide. Although a key step in the regulation of KLK function, details regarding the activation of the human pro-KLKs (i.e. the KLK "activome") are unknown, to a significant extent, but have been postulated to involve "activation cascades" with other KLKs and endopeptidases. To characterize more completely the KLK activome, we have expressed from Escherichia coli individual KLK propeptides fused to the amino terminus of a soluble carrier protein. The ability of 12 different mature KLKs to process the 15 different pro-KLK peptide sequences has been determined. Various autolytic and cross-activation relationships identified using this system have subsequently been characterized using recombinant pro-KLK proteins. The results demonstrate the potential for extensive KLK activation cascades and, when combined with available data for the tissue-specific expression of the KLK family, permit the construction of specific regulatory cascades. One such tissue-specific cascade is proposed for the central nervous system.

Highlights

KLK13 fusion protein with KLK11 resulted in the release of an aberrantly small propeptide because of hydrolysis between the P2 and P1 positions
The data generated in this study permit a comparative evaluation of the ability of the different KLKs to correctly process a particular KLK propeptide, and the data permit a comparative evaluation of the ability of a given KLK to process all 15 KLK propeptides
These results show that KLK11 is capable of dual substrate specificity and is in good agreement with the P1 specificity of KLK11 as determined using a positional scanning combinatorial library of tetrapeptides [52]

Summary

RESULTS

Purification of Pro-KLK Fusion Proteins—The pro-KLK fusion proteins were purified to apparent homogeneity, in each case, as determined by Coomassie Brilliant Blue-stained SDSPAGE (see supplemental figures). Coomassie Brilliant Blue-stained 16.5% Tricine SDS-PAGE analysis of 100:1 molar ratio incubation of pro-KLK-(1–15) (abbreviated as “Pro-K”) fusion proteins by mature KLK11 for 24 h at pH 7.4. Note, at this concentration the KLK11 control does not yield a stainable band. Summary of mass spectrometry and N-terminal sequencing data for the 24-h KLK11 hydrolysis of the pro-KLK-(1–15) fusion proteins at pH 7.4 (see Fig. 3 and text)

Theoretical mass

DISCUSSION

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