KLK14 (kallikrein-related peptidase 14)

	Figure 1. Schematic representation of the KLK14 gene. Exons are shown as boxes and introns as connecting lines. The coding sequences are highlighted as red, while 5' and 3' untranslated regions (UTRs) are shown in white. Numbers inside or outside boxes indicate lengths (nt) of exons and introns, respectively, while numbers in parentheses indicate lengths (aa) of protein isoforms. Arrows (↓) show the position of the start codons (ATG) and asterisks (*) denote the position of the stop codons (TAA or TGA). Question marks (?) indicate undetermined 5' UTRs. The figure is drawn to scale, except for the introns containing the (//) symbol.
Description	The KLK14 gene has a total length of 6,717 nt and consists of 8 exons and 7 intervening introns. The organization of the KLK14 gene is similar to that of the other KLK family members (Hooper et al., 2001; Yousef et al., 2001).
Transcription	The KLK14 pre-mRNA is subjected to alternative splicing, generating nine splice variants, four of which are accessible via the Reference Sequence (RefSeq) database. Three of them (GenBank accession numbers: NM_001311182.2, NM_022046.6, and NM_001369775.2) encode the main protein isoform (Kurlender et al., 2005). These three splice variants differ only in their 5' and 3'-untranslated regions (UTRs). The fourth transcript (GenBank accession number: NR_163144.1) lacks exon 5 and does not have an open reading frame (ORF). Three splice variants have been deposed in the Ensembl database, but only one of them shows a different exon combination, compared to the aforementioned transcripts; this transcript (Ensembl accession number: ENST00000391802.1) uses the same start codon as the predicted transcript and includes all other exons, leading to a protein isoform with distinct N-terminus, compared to the main isoform. Recently, members of our research group have discovered four novel KLK14 transcripts and submitted their sequences to GenBank. Two out of them possess an ORF and, most likely, generate two respective protein isoforms. The first transcript (GenBank accession number: KY305101.1) resembles the one available in Ensembl, but lacks two exons within the coding sequence, leading to a protein isoform with a distinct C-terminus compared to the main one; the second one (GenBank accession number: KY305102.1) lacks three exons within the coding region and uses alternative splice sites in its 3' coding region, thus probably generating a short peptide of 30 amino acid residues. The 5'UTR of both these transcripts has not been determined. The other two transcripts (GenBank accession numbers: KY305104.1 and KY305105.1) have an intron retention, do not have any ORF and, most likely, represent nonsense-mediated mRNA decay candidates. Another splice variant (GenBank accession number: XM_017026820.1) is predicted to be encoded by the KLK14 gene, based on automated sequence analysis of expressed sequence tags (ESTs). This splice variant probably uses a different translation start codon, lacks an exon within the coding sequence and uses alternative splice sites in its 3' coding region, in comparison with the aforementioned transcripts, generating a protein with distinct N- and C-termini, compared to the classical isoform precursor. Expression KLK14 mRNA is primarily expressed in the prostate, salivary gland, stomach, lung, spleen, uterus, thymus, liver, small intestine, and cerebellum while lower levels of expression are found in many other tissues. KLK14 mRNA expression is downregulated in malignant breast, testicular, prostatic, and ovarian tumors (Yousef et al., 2001).
Pseudogene	Not identified so far.

	Figure 2. Alignment of amino acid sequences of the precursors of the KLK14 protein isoforms. The three amino acid residues (positions: 67, 11, and 204) constituting the catalytic triad that is required for protease activity are shown in red. The N-terminal signal peptide (positions: 1-18) is shown in blue. Substrate binding sites (positions: 198, 219, and 221) are shown in light blue, while the cleavage site of the precursor form (position: 25) is indicated in orange. Numbering of amino acid residue positions is based on the sequence of the main KLK14 isoform (isoform 1) precursor.
Description	The precursor of the classical KLK14 isoform (isoform 1; GenBank accession numbers: NP_001298111.2, NP_001356704.1, and NP_071329.3) consists of 251 amino acid residues and has a molecular mass of 29.1 kDa. The N-terminal signal peptide comprises 18 amino acid residues. KLK14 is a secreted serine protease having dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues (Felber et al., 2005; Rajapaksei and Takahashi, 2007). KLK14 protein is synthesized as inactive precursor zymogen that is cleaved at the position 25 during limited proteolysis to generate its active form (Borgono et al., 2007). Three amino acid residues (positions: 67, 111, and 204) constitute the catalytic triad that is required for protease activity. Three other amino acid residues (positions 198, 219, and 221) constitute the substrate-binding site. A recent study has suggested some specific amino acid residues as responsible for the ability of KLK14 to interact with other proteins (Asp189, Ser190, Gln192, and Trp211) (Solis-Calero and Carvalho, 2017). KLK14 isoform 2 (Ensembl accession number: ENSP00000375678.1) consists of 267 amino acid residues and contains a distinct N-terminus, compared to isoform 1. KLK14 isoforms 3 and 4 (GenBank accession numbers: AQU42773.1 and AQU42774.1) share the same N-terminus as isoform 2 and consist of 125 and 30 amino acid residues, respectively. Additionally, another isoform, predicted to be generated by KLK14 splice variant X1 and composed of 179 amino acid residues, has distinct N- and C- termini, in comparison with the main KLK14 isoform precursor.
	Figure 3. Predicted models of the precursors of the KLK14 protein isoforms, using the I-TASSER server (Yang and Zhang, 2015). For each protein, only the 3D structure with the highest confidence score is presented. The RasMol 'Group' color scheme color codes residues by their position in a macromolecular chain. Each polypeptide is drawn as a smooth spectrum from blue through green, yellow and orange to red. Thus, the N-termini are colored blue and the C-termini are drawn in red.
Function	Several human protein substrates for KLK14 have already been identified, including major components of the extracellular matrix such as collagens I-IV, fibronectin 1, kininogen 1, fibrinogen, plasminogen (PLG), vitronectin (VTN), and insulin-like growth factor-binding proteins 2 and 3 (IGFBP2 and IGFBP3) (Borgono et al., 2007; Felber et al., 2005; Kryza et al., 2020). Thus, this secreted cancer-related peptidase may be involved in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage (Borgono et al., 2007). Additionally, KLK14 is implicated in other physiological functions too, such as desquamation and activation of signaling molecules associated with inflammation and cancer (de Veer et al., 2012). In fact, this enzyme may be responsible for as much as 50% of the total trypsin-like activity in stratum corneum, thus suggesting an important role for this very efficient protease under normal and disease conditions in the skin (Stefansson et al., 2006). KLK14 can also directly cleave semenogelins I and II (SEMG1 and SEMG2), thus playing a major role in seminal clot liquefaction (Emami et al., 2008). In addition, active KLK14 is efficiently able to cleave C3, the point of convergence of the complement cascade, indicating a potential modulation of C3-mediated functions. Thus, it parcipates in the activation of the innate immune response (Oikonomopoulou et al., 2013). Members of the proteinase-activated receptor (PAR) family constitute also targets of KLK14; their activation upon cleavage by KLK14 leads to differential signaling and affects tissue function (Ramachandran et al., 2012). For instance, KLK14 acts on proteinase-activated receptor 2 (F2RL1 or PAR2) to induce MAPK3/ MAPK1 (ERK1/ERK2) signaling pathway in colon cancer ID: 5006> cells (Gratio et al., 2011). Besides its other actions, human KLK14 activates the KLK proteolytic cascade (Emami and Diamandis, 2008). Very recently, it has also been suggested that elevated KLK14 activity contributes at multiple levels the activation of the oncogenic hepatocyte growth factor ( HGF)/MET signaling in prostate cancer and other human malignancies (Reid et al., 2016). Regarding the regulation of the enzymatic activity of KLK14, the serpins SERPINA1 (alpha-1 antitrypsin), SERPINF2 (alpha-2 antiplasmin), SERPINC1 (antithrombin III), SERPINA3 (alpha-1 antichymotrypsin) and SEPPINA12 (alpha-12), as well as SERPINE1 (plasminogen activator inhibitor-1) constitute inhibitors of this serine protease (Borgono et al., 2007; Felber et al., 2006; Ulbricht et al., 2018). Serine peptidase inhibitors, Kunitz type 1 SPINT1) and 2 (SPINT2), have been also shown to bind KLK14 (Solis-Calero and Carvalho, 2017). Moreover, citrate enhance KLK14 activity, whereas zinc ions exert inhibitory action on KLK14 (Borgono et al., 2007; Felber et al., 2006). Fragments of serine peptidase inhibitor Kazal type 5 (SPINK5; also known as LEKTI) specifically inhibit KLK14 as well as other KLKs, thus controlling desquamation through a pH-dependent interaction (Deraison et al., 2007). Moreover, transgenic mice bearing the human KLK14 gene and with Netherthon syndrome (SPINK5 deficiency) showed higher proteolytic activity in the granular layers and hair follicles, due to the higher KLK14 levels, as well as epidermal hyperproliferation and differentiation, downregulation of desmogleins 3 (DSG3) and 4 (DSG4), and IL36 family overexpression (Gouin et al., 2020).

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