Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

Abstract

Most kallikrein-related peptidases (KLKs) are N-glycosylated with N-acetylglucosamine 2 -mannose 9 units at Asn-Xaa-Ser/Thr sequons during protein synthesis and translocation into the endoplasmic reticulum. These N-glycans are modified in the Golgi machinery, where additional O-glycosylation at Ser and Thr takes place, before exocytotic release of the KLKs into the extracellular space. Sequons are present in all 15 members of the KLKs and comparative studies for KLKs from natural and recombinant sources elucidated some aspects of glycosylation. Although glycosylation of mammalian KLKs 1, 3, 4, 6, and 8 has been analyzed in great detail, e.g., by crystal structures, the respective function remains largely unclear. In some cases, altered enzymatic activity was observed for KLKs upon glycosylation. Remarkably, for KLK3/PSA, changes in the glycosylation pattern were observed in samples of benign prostatic hyperplasia and prostate cancer with respect to healthy individuals. Potential functions of KLK glycosylation in structural stabilization, protection against degradation, and activity modulation of substrate specificity can be deduced from a comparison with other glycosylated proteins and their regulation. According to the new concept of protein sectors, glycosylation distant from the active site might significantly influence the activity of proteases. Novel pharmacological approaches can exploit engineered glycans in the therapeutical context.

Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

“…As shown in the literature, KLK7 and the mutants produced in the Pichia system were not N-linked glycosylated (data not shown) due to the presence of a sequon at Asn239 in the terminal α-helix 24 . Therefore, the observed high molecular weight bands of stratum corneum-extracted KLK7 may be partially O-linked glycosylated at Thr144 as predicted by the NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc/), however not further verified to date 25 . Although we cannot exclude that difference in such complex post-translational regulation may have had some effect on the cleavage preference of KLK7, we note the selectivity profiles obtained here were similar to those obtained in other studies using mammalian cell-line derived KLK7 9 .…”

Kallikreins – The melting pot of activity and function

“…Depending on the glycosylation pattern, the activation cleavage results in release of two different active variants of KLK13. The presence of a glycan attached to Asn30 prevents cleavage after Phe35 [36]. The presence of glycans affects the activity of most kallikreins, including KLK-3, -4, -8, and -13.…”

“…The presence of glycans affects the activity of most kallikreins, including KLK-3, -4, -8, and -13. The exceptions are KLK-1, -2, and -15 [36]. However, research to date on kallikrein glycosylation has been conducted using proteins expressed either in bacterial or yeast expression systems, resulting in no glycosylation or non-homogeneous glycosylation.…”

Kallikrein-related peptidase 6 exacerbates disease in an autoimmune model of multiple sclerosis

“…Although more investigation will be needed to determine the significance of these unique Klk6 bands, it is possible that the 25 kDa band represents additional active protease, or its zymogen precursor, based on the expected molecular weight of the enzyme (Bernett et al, 2002; Blaber et al, 2002; Scarisbrick et al, 2012a,b). Based on descriptions by others, the 28 kDa band may be a differentially glycosylated form of Klk6 (Kuzmanov et al, 2009; Guo et al, 2014), but additional efforts will be needed to clarify this and its functional significance. As the pro-peptide for Klk6 contains only five additional amino acids that would add approximately 500 Da to its molecular weight, it is not possible to distinguish between active Klk6 and its zymogen precursor using Western blot alone.…”