Porcine kallikrein‐4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts

Abstract

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

Functions of KLK4 and MMP-20 in dental enamel formation

“…Pig and mouse both have three asparagine residues in the appropriate context for glycosylation, while human KLK4 has only one potential site. Following deglycosylation with glycopeptidase F, pig KLK4 converts into a single, inactive band with an apparent molecular weight of 28-kDa (Ryu et al, 2002). This observation seems inconsistent with recent findings that unglycosylated recombinant human KLK shows activity (Yoon et al, 2007).…”

“…Recombinant human KLK4 has also been shown to catalyze cleavages in multiple amino acid contexts (Matsumura et al, 2005;Obiezu et al, 2006), and among all of the kallikreins is the most active in cleaving the pro-sequence from the zymogens of other KLKs, but not its own (Yoon et al, 2007). MMP-20 (and thermolysin) can activate proKLK4 in vitro, but it is not clear if MMP-20 serves this function in vivo (Ryu et al, 2002).…”

“…KLK4 isolated from developing pig teeth cleaves amelogenin in vitro at numerous sites and in many different amino acid contexts (Ryu et al, 2002). KLK4 also digests a glycosylated enamelin cleavage product that is resistant to further digestion by MMP-20 (Yamakoshi et al, 2006).…”

Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales

“…MMP20 diverged from other matrix metalloproteinase loci prior to the common ancestry of tetrapods [24], and plays a key role in processing structural proteins (amelogenin, ameloblastin, enamelin) that are secreted into the extracellular matrix by ameloblasts during the secretory stage of enamel formation [23,25 -28]. MMP20 may also be necessary to activate kallikreinrelated peptidase 4 (KLK4; [29,30]), which cleaves and degrades remnants of enamel matrix proteins during the maturation stage of amelogenesis. MMP20-deficient mice have an amelogenesis imperfecta phenotype that is characterized by thin, hypomineralized enamel that easily chips away from the underlying dentin [31,32].…”

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

“…Murine mKLK4 possesses three sequons at Asn109, Asn159, and Asn202; it shares the latter two with porcine pKLK4 that has a third sequon at Asn120 (Figure 2). Recombinant pKLK4 from E. coli, insect cells and HEK293 cells, as well as natural samples from pig teeth displayed increasing molecular weights of 28 (eco), 31 (insect), 34 (HEK293), and 34 to 37 kDa (natural), indicating increasing degrees of glycosylation (Ryu et al, 2002). However, only natural pKLK4 was significantly active against small fluorogenic compounds and the physiological substrate amelogelin.…”