Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking

Abstract

MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein–protein and protein–lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.

Introduction

Matrix metalloprotease 23 (MMP23) belongs to a large family of zinc-dependent endopeptidases that function at neutral pH to degrade extracellular matrix proteins, cleave cell-surface receptors, release apoptotic ligands, and activate chemokines and cytokines [1–3]. The 23 MMPs in humans are encoded by 24 genes, with two identical genes on chromosome 1 encoding MMP23 (MMP23A and MMP23B) [4]. Matrix metalloproteases (MMPs) are involved in a wide range of cellular processes, including tissue remodeling, cell proliferation, cell migration, differentiation, angiogenesis, apoptosis, and the immune response [5–10]. Besides these normal physiological processes, MMPs have been implicated in a large number of pathological processes such as arthritis, Alzheimer’s disease, atherosclerosis, vascular disease, central nervous system disease, liver cirrhosis, and various cancers [11, 12].

MMP activity is regulated at a number of different levels, including biosynthesis (transcription/translation) [13], zymogen activation [11, 13], compartmentalization [11, 14, 15], and inactivation [16–19]. Some MMPs are required during development and normal physiology and may play a role in homeostasis [8, 10, 20–23], while others are produced in response to tissue injury and infection [5, 8]. In chronically inflamed tissues and most cancers, MMPs contribute to a number of pathological processes, including tissue degradation, tumor progression and invasion [24–31].

MMPs are synthesized as zymogens and usually secreted or anchored to the plasma membrane, confining their activity to the extracellular environment or the cell surface. Recent evidence suggests that secreted MMPs bind to specific cell-surface receptors, membrane-anchored proteins, and cell-associated extracellular matrix (ECM) molecules, and function pericellularly at focused locations [15]. MMPs are also found in the cell nucleus (MMP2, 3, 9, 13, and MT1-MMP), cytoplasm (MMP1, 2, 26, and 23), and various organelles, where their localization is mediated by interactions with other proteins, proteoglycan core proteins, and/or their glycosaminoglycan chains and other molecules [11, 14, 32–43].

This review focuses on the structural and functional features that distinguish MMP23 from other MMPs. MMP23 is a membrane-anchored protein consisting of 391 amino acid residues and four separate domains, three of which differ significantly from those of other MMPs. The N-terminal pro-domain of MMP23 lacks the enzymatic inhibitory sequence motif and folded globular structure characteristic of other MMP pro-domains, and has been shown to modulate voltage-gated potassium (Kv1) activity by regulating the intracellular trafficking of these channels. MMP23 also contains a toxin-like domain (TxD) that has been shown to block Kv1 channels. In addition, the C-terminal hemopexin domain common to other MMPs is replaced by an immunoglobulin-like cell adhesion molecule (IgCAM) domain of unknown function in MMP23.

Domain structure

MMPs typically contain a short signal sequence followed by a pro-domain, a central zinc- and calcium-dependent catalytic domain, a linker region (also called the ‘hinge region’), and a C-terminal hemopexin domain (Fig. 1) [11, 44–46]. MMP23, by contrast, contains an N-terminal pro-domain that includes a membrane-anchoring transmembrane domain (TMD), a cysteine-rich TxD located immediately after the catalytic domain, and a C-terminal IgCAM domain.

Fig. 1.

Schematic of the domain architecture of human matrix metalloproteases. Most MMPs contain a pro-domain (red), a catalytic domain (blue), a linker (hinge region, dark green) and a hemopexin domain (green). Furin-activated MMPs, including the membrane-anchored MMPs, possess a basic RX{K/R}R sequence motif at the C-terminal end of the pro-domain (where X represents any amino acid residue). Cleavage at this site results in release of the pro-domain and activation of the enzyme. Two MMPs (MMP2 and MMP9) contain three fibronectin (FnII)-like repeats (yellow) in the catalytic domain prior to the catalytic Zn²⁺ ion-binding site. Four MMPs (MT1, 2, 3, 5-MMP) are anchored to the cell membrane via a C-terminal type-I transmembrane domain (TMD) and two MMPs (MT4, 6-MMP) are tethered by a glycosylphosphatidylinositol (GPI)-anchor. In contrast, MMP23 is anchored via a N-terminal type-II transmembrane domain (gray). The two minimal domain MMPs (MMP7, 26) and MMP23 lack the hemopexin domain, and in MMP23 this domain is replaced by a C-terminal cysteine-rich toxin-like (TxD, brown) domain and an immunoglobulin-like cell adhesion molecule (IgCAM) domain (purple)

MMP23 is located in a duplicated region of human chromosome 1p36.3 (Fig. 2). The two MMP23 genes, referred to as mmp23a (pseudogene, MMP23A) and mmp23b (MMP23B denoted as MMP23 in this review), each produce three alternatively spliced products, MMP23, MMP23a, and MMP23b (also known as MMP21/22A, MMP21/22B, and MMP21/22C, respectively) (Fig. 2). Rodent genomes contain a single copy of the MMP23 gene, implying that the two copies in humans evolved from a recent duplication event that occurred after the divergence of the rodent and primate linages [47]. Comparative genomics analyses indicate that the TxD and IgCAM domains were introduced during the early evolution of tetrapods, since zebrafish has an orthologue of this gene that lacks these C-terminal domains [48].

Fig. 2.

Structure of the human MMP23 loci and the corresponding gene product [4]. Schematic representation of the ~150-kb region of chromosome 1p36.3 that contains the two metalloprotease 23 genes (mmp23a and mmp23b). The genes are represented by arrows orientated to highlight their transcriptional orientation relative to the neighboring CDK11 (cyclin-dependent kinase 11), MIB2 (mindbomb E3 ubiquitin protein ligase 2), and SLC35E2B (solute carrier family 35, member E2B) genes. The exon/intron structure and protein domain architecture for the MMP23 gene and isoforms (MMP23, MMP23a, and MMP23b) formed by alternative splicing are also illustrated. The location of exons constituting the mmp23 gene and protein (MMP23) are listed in the table

Pro-domain

Most MMPs are expressed as zymogens (inactive enzymes) where the N-terminal pro-domain adopts a folded globular structure that interacts with the catalytic domain and maintains the enzyme in a latent state (Fig. 3). The highly conserved pro-domain possesses a ‘cysteine-switch’ motif (PRCGXPD) containing a cysteine residue that coordinates with the catalytic zinc ion (Fig. 3b, c) [49]. Destabilization or removal of the pro-domain unblocks the active site, allowing it to process substrate [46]. Protease activation can occur either by direct cleavage of the pro-domain by itself (autolysis) or by another protease, or by modification of the cysteine that binds to the catalytic zinc ion [11, 49, 50].

Fig. 3.

MMP pro- and catalytic domain structures. a Superposition of the catalytic (blue) and pro- (red) domains of MMP1 (PDB id: 1SU3), MMP2 (1CK7), MMP3 (1SLM), MMP9 (1L6J). The fibronectin-related domains in MMP2 and MMP9 have been removed for clarity. Zinc and calcium ions are colored magenta and green, respectively. b Pro- and catalytic domains of MMP3 where residues of the conserved ‘cysteine-switch’ (PRCGXPD, where X is any amino acid residue) are colored yellow. c Magnification of (b) where side chains for residues comprising the ‘cysteine-switch’ motif are labeled. The figure was generated using the program Pymol [173]

The 80-residue MMP23 pro-domain sequence, by contrast, is shorter and significantly different from those of other MMP pro-domains. It lacks the classical cysteine-switch motif and displays only limited sequence homology among different species [51, 52]. Rather than the folded globular structure formed by other MMP pro-domains, the MMP23 pro-domain contains a N-terminal type-II TMD (₂₀WLGAALVALCLLPALVLL₃₇) that anchors the protein to the membrane [39, 51–54]. In contrast, other membrane-tethered MMPs (MT-MMPs) are anchored to the membrane by a C-terminal TMD (MMP14, 15, 16, and 24) or a glycosylphophatidylinositol (GPI) anchor (MMP17 and 25) [55, 56]. The MMP23 TMD acts as a signal peptide that directs MMP23 to the ER/Golgi prior to secretion [7, 9–11], in a manner similar to the N-terminal cleavable signal peptide of other MMPs, which is absent in MMP23 [54].

The MMP23 TMD sequence displays only limited homology across various species, although there is strong conservation of several hydrophobic residues (Fig. 4a). Helical wheel analysis of residues in the MMP23 TMD shows that these conserved residues cluster in two regions on the surface of this transmembrane helix (Fig. 4b), suggesting that they may play a functional role [38].

Fig. 4.

Conservation of residues within the TMD of the pro-domain of MMP23. a Sequence alignment of pro-domain regions of MMP23 from various species where residues comprising the TMD are colored red, the furin recognition motif is green and the catalytic domain is gray. The accession numbers for each MMP23 are: human, UniProt 075900; mouse, 088676; rat, 088272; monkey, 10FJ41; cow, Q2TBM7; pig, F1RJC6; dog, E2R4V4; panda, G1M504; chicken, E1BX58; and possum, F7CDE4. Residues corresponding to the conserved astacin FXGD motif are colored blue. The sequence alignment was performed using ClustalW [174]. Residues are numbered according to human MMP23. The level of conservation is illustrated along the lower row: Asterisk identical, colon highly conserved, dot weakly conserved. b Helical wheel representation of the alignment of the TMD in (a) generated using the web-based program WHEEL (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Identical and conserved residues are denoted by X and residues that cluster on separate faces of the helical wheel are denoted by a dotted blue line. Residues are represented as follows: hydrophilic—circles, hydrophobic—diamonds, and potentially positively charged—pentagons. Residues are color-coded according to hydrophobicity/hydrophilicity, from red (most hydrophilic) through yellow (moderately hydrophilic) to green (most hydrophobic). Potentially charged residues are colored light blue. c Secondary structure of pro-domain for human MMP23. Model of the TMD (α1 helix) and juxta-membrane helix (α2 helix) of the pro-domain for human MMP23 in a lipid membrane (residues 1–74). Atoms of the side-chain groups of residues in the two helices are highlighted. Phosphate groups of the lipid are represented as blue spheres; lipid choline, glycerol and acyl chains, and water are omitted for clarity. The amino acid sequence of the MMP23 pro-domain is shown at the top where α-helical regions are boxed and residues of the TMD are colored red. Adapted from Nguyen et al. [38]

NMR studies of the MMP23 pro-domain in dodecylphosphocholine (DPC) micelles show that it contains a relatively long α-helix (α1) (residues Glu17–Leu40), incorporating the membrane-anchoring TMD, connected by a 6-residue linker to a short juxta-membrane α-helix (α2) (residues Ala47–Leu58) (Fig. 4c) [38]. Paramagnetic relaxation enhancement and solvent exchange measurements indicate that the N-terminal capping region of helix α1 (residues Glu17–Arg19) is exposed to solvent while the remaining portion of the helix is buried within the DPC micelles and not accessible to solvent [38]. The juxta-membrane helix (α2) appears to adopt a partially flexible structure that interacts with the surface of the micelle and is partially buried [38].

The short N-terminal cytoplasmic tail of the MMP23 pro-domain is unstructured [38], and it is not known whether this region plays a regulatory role as in other MT-MMPs. MT-MMPs can modulate intracellular processes through interactions of their short unstructured cytoplasmic C-terminal tail with various intracellular proteins, leading to the induction of intracellular signaling pathways [57, 58]. Most of these interactions occur at the plasma membrane but they can also take place in other organelles such as the Golgi [58–66].

Some MMPs, including all MT-MMPs, contain a furin-recognition motif between the pro- and catalytic domains (Figs. 1, 4) [13, 50]. Cleavage at this site releases the inhibitory pro-domain and activates the enzyme in the secretory pathway [50]. MMP23 also contains a furin recognition motif ₇₆RRRRY₈₀ between the propeptide and catalytic domain [52, 54], and cleavage between residues Arg79 and Tyr80 has been shown to release the active enzyme [67–70]. However, it is not known whether furin is the principal protease involved in the activation of MMP23 [51].

The pro-domain of MMP23 differs significantly from those of other MMPs, and it is unclear how it is maintained in a latent state during passage through the secretory pathway. The MMP23 pro-domain contains a poly-proline motif immediately prior to the furin recognition motif (i.e. ₆₈RVPGPLAPRRRRY₈₀) that exhibits limited similarity to various SH3 domain ligands [71, 72]. This motif may bind to the catalytic active site and inhibit activity, although this region is poorly conserved across species.

MMPs generally display many similarities with the astacins including (1) structural homology between the MMP catalytic domain and upper N-terminal subdomain (NTS) of the astacins (Fig. 5a, b), (2) similar zinc-binding motifs, (3) a partially unstructured N-terminal pro-segment, and (4), in several astacins, the presence of one or more toxin-like (ShK-like) domains similar to the TxD of MMP23 [73–75]. The MMP23 pro-domain displays several characteristics similar to the pro-segment of astacin zinc-dependent metalloproteases (Fig. 5) [73]. The astacin pro-segment is variable in length (34 residues for crayfish astacin to 393 residues for the Drosophila tolloid-related 1 protease) [76] and contains only a short FXGD consensus sequence [73, 76]. The aspartate residue within this FXGD motif binds the catalytic zinc and inhibits protease activity via an ‘aspartate-switch’ mechanism. The structure of crayfish pro-astacin shows that the pro-segment extends across the front surface of the enzyme in the opposite direction to that of the substrate, with a short helical motif located towards the N-terminus (Fig. 5c). MMP23 contains a short sequence (₅₁AQGD₅₄; Fig. 4a), similar to the FXGD motif of the astacins, which is located within the juxta-membrane helix ₄₇AWSAAQGDVAAL₅₈ of the MMP23 pro-domain. However, this motif varies among MMP23s from different species (Fig. 4a), suggesting that the pro-domain may inhibit the catalytic domain via a novel mechanism.

Fig. 5.

Structure of pro-astacin. a Two orthogonal views of a superposition of the X-ray crystal structures of the activated catalytic domains of crayfish astacin (PDB ID: 1AST, orange) and MMP3 (1SLM, blue). The N- (NTS) and C-terminal (CTS) subdomains of crayfish astacin are denoted by open circles. b Crystal structure for pro-astacin (3LQ0) depicting the backbone of the pro-segment as sticks (green) and the enzyme moiety as a ribbon (orange). c Magnification of (b) where side-chains for residues comprising the conserved FXGD motif are shown (yellow) and labeled while the catalytic zinc ion is illustrated as a magenta sphere

Catalytic domain

MMP catalytic domains, including MMP23, share a high degree of structural (Fig. 3a) and sequence homology (Fig. 6a) [77–79]. The MMP23 catalytic domain structure has not yet been determined, although it is likely to be similar to that for other MMPs since they share such a high degree of sequence and structural homology.

Fig. 6.

MMP catalytic domain. a Sequence alignment of the catalytic domains of MMP23 with soluble MMPs (MMP1, UniProt P03956; MMP3, P08254; MMP7, P09237; MMP8, P22894; MMP10, P09238; MMP11, P24347; MMP12, P39900; MMP13, P45452; MMP19, Q99542; MMP20, O60882; MMP21, Q8N119; MMP27, Q9H306; MMP28, Q9H239), MT-MMPs (MMP14, P50281; MMP15, P51511; MMP16, P51512; MMP17, Q9ULZ9; MMP23, O75900; MMP24, Q9Y5R2; MMP25, Q9NPA2) and fibronectin-like domain containing MMPs (MMP2, P08253; MMP9, P14780). Residues binding to calcium (C1 and C2) and zinc (Z1 and Z2) ions are colored blue and red, respectively, while those located in the specificity loop (SL) are colored green. The conserved Met residue of the ‘Met-turn’ (MetT) is colored purple, and the conserved Asp or Glu residues involved in stabilizing electrostatic interactions (NA) are brown. Secondary structure elements based on the X-ray crystal structure of human MMP1 (PDB ID: 1SU3) are shown above the sequence alignment where arrows represent β-strands and cylinders are α-helices. b, c MMP catalytic domain structure. b Catalytic domain of MMP8 (1JAN) showing secondary structure elements (orange arrows for β-strands, β1-βV; dark green helices for α-helices, αA-αC) and the four metal ions (zinc ions are colored magenta and calcium ions are light green). Side-chains for residues binding the catalytic zinc ion and forming salt-bridges that stabilize the Met-turn and N-terminus are illustrated with yellow carbon atoms and labeled for MMP8 and MMP23 (in brackets). The Met-turn is colored blue and the specificity loop is red. c Expanded view of active site region depicting side-chains for residues engaged in zinc binding and residues forming the specificity pocket (labeled). The figure was prepared using the program Pymol [173]. d Two orthogonal views of the crystal structure (PDB ID: 1UEA) of MMP3 (blue) bound to TIMP1 (orange)

MMP catalytic domains possess a globular conformation with a diameter of ~40 Å and a shallow active site cleft that binds substrates in an approximately extended conformation [78]. They have a common architecture comprised of three α-helices and a five-stranded β-sheet, and typically contain four metal ions (two calcium and two zinc) in highly conserved calcium- and zinc-binding sites (Fig. 6b, c) [44, 77, 78]. The extended zinc-binding consensus sequence, HEXXHXXGXXH (where X represents any residue) [80, 81], ‘Met-turn’ and, N-terminal anchoring residues typically found in MMPs are conserved in MMP23, while the ‘specificity loop’ that contributes to substrate specificity is significantly different (Fig. 6a, b).

The MMP23 catalytic domain contains two inserts that are not found in other MMPs (Fig. 7a). The first is located in the loop between strands βIII and βIV and is flanked by two Cys residues (₁₅₃CLVSAVHHC₁₆₁) that may form a disulfide bond [52]. Residues on either side of this loop interact with the structural zinc ion and one of the calcium ions, and it is possible that a disulfide bond within this loop may help to restrain these residues so that they can ligate these metal ions. The second insert (₁₉₃TRYSWKKGV₂₀₁) is positively charged and lies in the loop between strand βV and helix αB, which contains the fibronectin type II repeats that are important for substrate recognition in MMP2 and MMP9 (Fig. 7a) [52, 82]. Interestingly, the MMP23 isoform MMP23a contains a 41-residue insert at this site although the insert displays no sequence homology to the fibronectin type II domain (Fig. 7).

Fig. 7.

MMP23 isoforms. a Schematic of domain architecture of MMP23 showing the sequence and location of inserted sequences for isoforms MMP23a and MMP23b. b Sequence alignment for various human MMP23 isoforms (MMP23, O75900; MMP23a, O75900-2; MMP23b, O75900-3). Residues corresponding to the pro-domain are colored gray (TMD colored black), catalytic domain are blue, TxD are brown, linker domain are green and IgCAM domain are purple. Residues of the insert between the catalytic domain and TxD in the MMP23 isoform MMP23b are colored orange

MMP proteolytic activities are tightly regulated by several endogenous inhibitors since uncontrolled proteolytic activity following activation would result in tissue damage and functional alterations. Plasma-associated MMPs are inhibited by liver-secreted α₂-macroglobulin, while tissue or extracellular MMPs are regulated by a family of four tissue inhibitors of metalloproteases (TIMP1-4) [7, 13, 16, 17, 83]. Human α₂-macroglobulin is a tetrameric 725-kDa glycoprotein that entraps MMPs via a ‘venus flytrap’-like mechanism [84]; this bound complex is rapidly cleared by receptor (low density lipoprotein receptor-related protein-1)-mediated endocytosis [85, 86]. The in vitro proteolytic activity of MMP23 is inhibited by the tissue inhibitors of metalloproteases TIMP1 [54] and TIMP2 [52]. TIMPs (TIMP1–4) are comprised of 184–194 amino acid residues that form structured N- and C-domains that are stabilized by six disulfide bonds. The TIMP N-terminal domain binds tightly to the MMP catalytic domain, blocking the substrate binding site and inhibiting protease activity (Fig. 6d) [19, 87].

Immunoglobulin-like cell adhesion molecule (IgCAM) domain

The C-terminal immunoglobulin-like cell adhesion molecule (IgCAM) domain of MMP23 is unique among the MMPs. All other MMPs either possess a hemopexin C-domain or contain no C-terminal domain (Fig. 1) [7, 44, 46]. The hemopexin domain acts as a secondary specificity site or “exosite” located outside the active site, which binds and helps to correctly orientate the substrate [88]. This additional protein-binding domain increases the affinity of the protease for the substrate and can modify the specificity of the MMP catalytic domain [89, 90]. Exosites can also play a role in preparing substrates [91] such as localized unwinding of triple helical native collagen prior to cleavage [92]. The C-terminal immunoglobulin-like cell adhesion molecule (IgCAM) domain of MMP23 may also mediate protein–protein interactions similar to the hemopexin domain of other MMPs.

The MMP23 IgCAM domain displays sequence similarity to the Ig-like C2 type domains of the immunoglobulin superfamily (Fig. 8a, b). These domains mediate protein–protein interactions among themselves and with other types of molecules such as components of the ECM, signaling molecules, and several membrane-bound proteins (e.g., growth-factor receptors, integrins and cadherins) [93–97], as well as mediating direct membrane interactions (e.g., perforin) [98–100]. Protein or lipid interactions mediated by the MMP23 IgCAM domain may help to target and position protein substrates for proteolytic cleavage, similar to the hemopexin domain of other MMPs.

Fig. 8.

The Ig-CAM domain of human MMP23 shares sequence homology with the Ig-CAM and Ig-like C2 type domains from various proteins. Phylogenetic (PHYLIP) [175] analysis (a) and sequence alignment (b) of the IgCAM domains of human MMP23 (UniProt 075900) with rat CDON (XP_003751100), human Brother of CDO (Q9BWV1), human ROBO1-4 (ROBO1, Q1RMC7; ROBO2,Q9HCK4; ROBO3, Q96MS0; ROBO4, Q8WZ75), dog hemicentin (XP_548414), and human NCAM1 (P13591) and NCAM2 (O15394). Residues identical to human MMP23 are colored red, conserved residues are blue, and cysteine residues involved in disulphide bonds are green. Secondary structural elements based on the X-ray crystal structure of human ROBO1 (PDB ID: 2V9R) are shown above the sequence alignment where arrows represent β-strands and cylinders are α-helices. Adapted from Rangaraju et al. [39]

Toxin-like domain (MMP23-TxD)

The MMP23 toxin-like domain (MMP23-TxD), which lies immediately after the catalytic domain and is joined to the C-terminal IgCAM domain by a short Pro-rich linker (Fig. 7), is highly conserved from humans to hydra (Fig. 9a) [39]. The MMP23b isoform of MMP23 contains a short 25-residue Gly/Pro-rich linker joining the catalytic and MMP23-TxD domains that is not found in the other two isoforms (Fig. 7). The MMP23-TxD domain shares homology with the ShKT family of proteins from worms, cnidarians, and plants as well as the ion channel regulatory (ICR) domains of CRISPs (Fig. 9b). Phylogenetic analysis places the MMP23 TxDs, the sea anemone toxins, and the ICR-CRISP domains in distinct but related clades (Fig. 9c). MMP23 TxD is also closely related to ShKT domains in MFAP2 [101], Caenorhabditis elegans proteins (astacin metalloprotease NAS14, tyrosinase Try3, ligand-gated channel Igc22 and Mab7) [102], hydra and jellyfish astacin metalloproteases (HMP2 and PMP1) [103, 104], and plant oxidoreductases [2OG-Fe(II)] and propyl-4-hydroxylases (Fig. 9c). The TxDs in MMP23 and the ICR domains in CRISPs are encoded by a single exon, suggesting that these domains originate from an ancient exon. MMP23-TxD is most similar to the sea anemone toxin BgK, with identical or equivalent residues at 14 of 36 positions [39, 105–107]. The solution structure of a synthetic 37-residue peptide corresponding to rat MMP23-TxD also shows close similarity to the sea anemone toxins BgK [105] and ShK [107] (Fig. 9d) [39]. Rat MMP23-TxD possesses three short α-helices encompassing residues 10–14, 23–29, and 31–34, and a salt-bridge between Asp5, which is conserved in the ShKT domain family, and Arg32 (Ser in human MMP23 and Lys in ShK and BgK) that has been shown to be important for the correct folding of several sea anemone toxins [107–109]. Other MMP-TxDs contain conserved Ser or Thr residues at positions 32 and 33 (Fig. 9b) and may form a hydrogen bond to the side chain hydroxyl or the peptide backbone of either of these residues. MMP23-TxD shares greater structural similarity with BgK than ShK (r.m.s. deviation of backbone heavy atoms of 2.28 and 2.77 Å, respectively) (Fig. 9d). MMP23-TxD, BgK, and ShK have a turn involving the fifth Cys residue that is followed by a short α-helix (residues 31–34) in MMP23-TxD and BgK but not ShK [39]. The main differences between MMP23-TxD and BgK are in the length of the first two helices, with the first helix of MMP23-TxD being shorter and the second longer than in BgK.

Fig. 9.

The toxin-like domain (TxD) of MMP23 is conserved across species and is similar to the cysteine-rich ShKT family of proteins and the ion channel regulatory (ICR) domains of CRISPS. a Multiple sequence alignment of MMP23 TxDs from diverse species. Human (UniProt O75900), monkey (I0FJ41), pig (B0FXJ8), cow (Q2TBM7), dog (E2R4V4), rat (O88272), mouse (O88676), ferret (M3YSR1), opossum (F7CDE4), chicken (E1BX58), zebra finch (H0YXW0), xenopus (F6RIT9), turtle (K7FBH1), stickleback fish (G3NNG1), puffer fish (H3CZY7), zebra fish (F1R0R0), lancelet (C3Y5G9), C. elegans (Q9XTD6), sea urchin (H3IL27), Nematostella vectensis (starlet sea anemone, A7RW59), and hydra (Q9XZG0). Arrows point to Asp5, Ser32 and Ser33. b Multiple sequence alignment of representative ShKT domains with the ICR domains of CRISPs. Residues identical to human MMP23-TXD are colored red, conserved residues are blue and cysteine residues involved in disulphide bonds are yellow. Sea anemone toxins include BgK (UniProt P29186), ShK (P29187), AeTX-K (Q0EAE5), AsKS (Q9TWG1), AeK (P81897), HmKT (O16846). Nas14-C. elegans, nematode astacin metalloprotease NAS14 (Q19269). Tyr3-C. elegans, tyrosinase 3 (Q19673). LGC-22-C. elegans, ligand-gated channel 22 (NP_500538). HMP2-Hydra, Hydra metalloprotease 2 (AAD33860). MFAP2-human, microfibril associated protein 2 (P55001). MAB-7, male abnormal protein 7 (NP_508174). PMP1-Jellyfish, Podocoryne metalloprotease 1 [103]. Arrows point to the conserved Lys/aromatic residue dyad found in many Kv channel-blocking toxins and Lys residue that is important for correct folding of several sea anemone toxins. c Evolutionary relationship of MMP23 TxD with sea anemone toxins, ShKT domains and ion channel regulatory (ICR) domains of cysteine-rich secretory proteins (CRISPs). The phylogenetic tree (PHYLIP) [175] was generated using the alignment in (c) and sequences for oxidoreductase, 2OG-Fe(II) oxygenase family protein from A. thaliana (NP_189490) and propyl-4-hydroxylase α-subunit, O. sativa japonica group (AAT77286). d Comparison of the solution structure of the MMP23 TxD (PDB id: 2K72) with those for sea anemone Kv channel blockers BgK (1BGK) and ShK (1ROO) where α-helices are colored red and loops are green. Disulfide bonds are colored yellow and the pore-occluding residue is blue and labeled. e Structure of ShK docked to a model of the pore vestibule region of Kv1.3 [108]. Kv1.3 is represented as a light brown ribbon, while ShK is shown as a molecular surface with all residues colored red except Lys22 (blue) which occludes the channel pore. Adapted from Rangaraju et al. [39]

BgK and ShK are potent voltage-gated potassium (Kv) channel blockers, which contain a positively-charged Lys residue (Lys25 in BgK, Lys22 in ShK) that occludes the channel pore (Fig. 9d, e) [110]. MMP23-TxD contains an evolutionarily conserved, similarly charged residue at this position (Arg276) and also blocks several Kv channels (Kv 1.1, 1.3, 1.4, 1.6, and 3.2) with IC₅₀ values in the nanomolar to low micromolar range while having no effect on others (Kv1.2, 1.5, 1.7, and KCa3.1) [39]. MMP23-TxD blocked human Kv1.6 channels expressed in COS7 cells with an IC₅₀ of 370 nM and Kv1.3 expressed in L929 fibroblasts and T cells with an IC₅₀ of 2.7 μM, compared to 10–20 pM [108, 111] for ShK [39, 108, 109, 112, 113]. The critical Lys/aromatic residue dyad found in many potassium channel-blocking toxins [114] is missing in MMP23-TxD (Fig. 9b), which may help explain its lower potency, although mutation of the corresponding residues in MMP23-TxD (Arg24/Leu25) to the conserved dyad motif (Lys/Tyr) did not improve potency against Kv1.3 (IC₅₀ of 2.7 μM) [39]. Previous studies have demonstrated that replacement of Lys22 in ShK with Ala abolishes channel blocking activity while replacement with shorter or longer positively charged residues retains activity, albeit with a lower potency [108, 109, 112, 113].

Potassium channel regulation of Kv1.3 channel trafficking

TxD-containing proteins may play a role in regulating the intracellular trafficking of Kv1 channels [115–117]. It has been suggested that the preponderance of intracellular Kv1 channels in many tissue types may be due to the interaction of a protein containing a TxD with Kv1 channels, retaining them within the ER [115–117]. This was supported by evidence showing that heterologous over-expression of ER-luminal dendrotoxin (DTX), which presumably competes with this TxD-containing protein, allows these channels to escape the ER and migrate to the cell surface [116]. This resulted in increased levels of cell-surface Kv1 channel, and patch-clamp analyses showed increased Kv1 current amplitudes, suggesting that the DTX does not remain bound during passage to the cell surface [116]. Recent evidence suggests that the TxD-containing protein may be MMP23 [39].

Full-length MMP23, which is found predominantly in perinuclear and ER membranes in mammalian cells [51, 53], has been shown to co-localize with Kv1.3, but not the closely-related Kv1.2 channels, in these organelles (Fig. 10c, d) [38, 39]. This specificity suggests that the observed attenuation in potassium channel activity may result from intracellular retention of the channels upon interaction with MMP23 (Fig. 10a, b) [38, 39].

Fig. 10.

The pro-domain of MMP23 specifically interacts with Kv1.3 and not the closely-related Kv1.2 channel. a, b Whole cell patch-clamp currents showing dose-dependent block of Kv1.2 (a) and Kv1.3 (b) by full-length MMP23 (MMP23-FL) and the pro-domain of MMP23 (MMP23-PD). c, d DsRed-MMP23-FL (full-length) and DsRed-MMP23-PD (pro-domain of MMP23) co-localise with Kv1.3-GFP (d) but not Kv1.2-GFP (c). Adapted from Nguyen et al. [38]. e, f Structural elements involved in Kv channel trafficking. e Sequence alignment of the S5 to C-terminal regions of Kv1.2 (UniProt P16389) and Kv1.3 (P22001) required for the intracellular trapping of Kv channels. Residues corresponding to the S5 and S6 transmembrane helices and specificity filter (SF) are highlighted by an orange rectangle above the sequences while the acidic and PDZ trafficking determinant (TD) motifs are denoted by red rectangles. Sequence alignments were performed using the program ClustalW [174]. f Cartoon representation of a Kv channel where the voltage-sensor domain (VSD) and pore domain TM helices are colored green and orange, respectively. Various TDs are denoted by red boxes. TDs include acidic and VXXSL (where X is any residue) motifs in the unstructured C-terminal tail of the Kv channel [118, 119], an ER retention TD in the turret domain external to the channel pore [115] and a motif in the extracellular loop between the TM helices S1 and S2 [123]. g Cartoon model of the assembled Kv channel where TDs of the C-terminal tail are denoted by red boxes

It was assumed initially that the intracellular retention of Kv1.3 channels was mediated by the MMP23-TxD [39]. However, truncation analyses showed that the pro-domain of MMP23 (MMP23-PD) alone could suppress Kv1.3, although not Kv1.2, currents to the same extent as the full-length protein (Fig. 10a, b) [38]. MMP23-PD and Kv1.3 were shown to be co-localized predominantly in the ER of COS-7 cells, similar to the full-length protein, indicating that the pro-domain of MMP23 mediates the intracellular trapping of Kv1.3 channels (Fig. 10c, d) [38].

Examination of the interaction of MMP23-PD with several Kv1.2/Kv1.3 chimeras showed that the Kv1.3 region from the S5 transmembrane helix to the C-terminal end of the channel is required for MMP23-PD-dependent channel modulation [38]. However, it remains to be determined whether this is mediated by a direct interaction of MMP23-PD with the channel. The sequences of the S5 and S6 transmembrane helices and selectivity filter for Kv1.2 and Kv1.3 are highly homologous, while the C-terminal tail differs between these proteins (Fig. 10e). The unstructured C-terminal tail contains several trafficking determinant (TD) motifs that have been shown to regulate the localization of Kv channels (Fig. 10f, g). These TD motifs include acidic and VXXSL motifs [118, 119] and a PDZ-binding motif at the end of the C-terminal tail that binds to the postsynaptic density 95 protein (PSD95) [120–122]. Other TD motifs include an ER retention TD in the turret domain external to the channel pore [115] and a motif in the extracellular loop between S1 and S2 [123].

Possible mechanisms for the modulation of ion channel activity

Intra- or extracellular helical segments adjacent to TMDs, similar to the juxta-membrane helix α2 (Ala47–Leu58) in the MMP23 pro-domain, are not uncommon [124, 125] and often play a functional role [126]. KCNE1, another potassium channel-modulating protein, contains an α-helical trans-membrane domain linked to two membrane-interacting juxta-membrane helical domains [125]. KCNE1 and the related KCNE2 protein both suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels by trapping them early in the secretory pathway [127, 128]. Furthermore, the closely-related protein KCNE4 suppresses Kv1.3 currents by inducing partial ER retention of the channel [129]. KCNE1 has additional channel-modulating effects on Kv7.1 channels mediated by the C-terminal juxta-membrane helical domain, which is not present in MMP23-PD [130]. These effects are mediated by a direct interaction of the TMD and C-terminal juxta-membrane helical domains of KCNE1 with the Kv channel or other accessory proteins [131]. It is possible that the TMD within MMP23-PD mediates a similar interaction with Kv1.3 channels but this remains to be confirmed.

Proteolytic cleavage by MMP23 may also regulate Kv channel activity. Protease-mediated regulation of ion channel activity can occur either directly by proteolytic cleavage and activation (or inactivation) of the channel [e.g., serine protease cleavage of epithelial sodium channels (ENaC)] [132, 133] or indirectly via cleavage of an upstream regulator of channel activity [e.g., serine protease cleavage of protease-activated receptor 2 (PAR₂) leads to the phosphorylation and activation of transient receptor potential (TRP) ion channels] [134, 135].

MMP23 shares several features with voltage-gated sodium channel β1 subunits (Na_Vβ1). Na_vβ1 subunits contain an N-terminal extracellular Ig-like domain, a single helical type-I transmembrane domain that anchors the protein to the plasma membrane and a C-terminal flexible intracellular domain [136]. Na_Vβ1 subunits normally form an integral component of Na_V channels and can act as cell adhesion molecules and modulate the cell surface expression of Na_V channels, enhancing sodium channel density and cell excitability [137]. However, Na_Vβ1 subunits can also co-assemble with and modulate the biophysical properties of Kv1 and Kv7 channels, although not Kv3 channels [138]. The extracellular Ig domain of Na_Vβ1 was required for the acceleration of Kv1.3 channel activation while the full-length protein was required for acceleration of Kv1.2 activity and slowing of Kv1.1 deactivation [138]. These interactions were mediated by an association of the Na_Vβ1 TM domain with the Kv1 channel pore and voltage-sensor domains, while the Ig domain forms extensive interactions with the S1–S2, S5–P and P–S6 extracellular loops of the channel [138].

Biological function

MMP23 and Kv1.3 exhibit overlapping tissue expression in many tissues where they may interact: lung, heart, uterus, placenta, ovary, testis seminiferous tubules, prostate, intestine, pancreatic islets, cingulated cortex, adrenal cortex, osteoblasts, chondroblasts, cartilage, synovium, natural killer cells, dendritic cells, and tendons [39, 51, 54, 139–146] [see BioGPS Web site (http://biogps.org)].

The physiological function of MMP23 is unknown, although there is evidence it may play a role in various physiological processes. Human MMP23 is expressed predominantly in ovarian, testis and prostate tissues [54]. MMP23 mRNA is also expressed in chrondrocytes and osteoblasts, implying involvement in cartilage and bone formation [139]. ADAM12 and MMP23 have also been linked to inflammatory disorders as they are co-expressed in painful tendinopathy [147]. Human MMP23 is also expressed in the synovium and cartilage [140], and in cranial sutures [148] and human aminochorion [141]. Expression of rat MMP23 occurs mainly in heart, spleen, and lung [52], while expression is spatially and temporally regulated in a cell-type-specific manner during follicular development [51]. In addition, porcine MMP23B was strongly expressed in ovaries and heart [149]. Differential expression of human MMP23 has been reported in breast [142] and prostate [150] cancers, as well as in multiple myelomas [151] and synovial sarcomas [152].

Microarray studies using a zebrafish liver cancer model have shown that mmp23b is significantly down-regulated in liver cancer, whereas other MMPs are up-regulated [153]. More recently it was reported that mmp23b is expressed in hepatocytes during normal development in zebrafish and that mmp23b deficiency leads to a reduction in liver size due to defective hepatocyte proliferation [154]. There is evidence that MMP23B functions through the tumor necrosis factor (TNF) signaling pathway [154]. TNF is a 26-kDa membrane-anchored pro-inflammatory cytokine that is a key regulator of liver homeostasis in mammals [155]. Hepatic TNF is derived mainly from Kupffer cells and regulates liver homeostasis by controlling hepatocyte proliferation and cell death [156, 157]. MMP23B directly interacts with TNF and mediates its release (shedding) from the cell membrane [154]. In vitro immunoprecipitation experiments have demonstrated that human MMP23B binds to human TNF, although it is not known if MMP23B cleaves TNF or whether this is mediated by another protein such as TACE/ADAM17 [TNF-converting enzyme transarterial chemoembolization, a member of the disintegrin family of metalloproteases (ADAMs)] [158].

We have recently shown expression of MMP23 and Kv1.3 in normal colonic epithelium with overlapping staining of both proteins in human colorectal cancers [38]. MMP23-PD may regulate the surface expression of Kv1.3 channels in primary and metastatic colorectal cancer cells and thereby regulate cellular function. Colon cancer cells secreting processed active MMP23 enzyme containing the Kv1.3 channel-blocking toxin domain (TxD) may block Kv1.3 channels on infiltrating anti-tumor T cells [38], and suppress them as a means of immune evasion. Tumor cells would be protected from the MMP23 TxD because their Kv1.3 channels are trapped intracellularly upon interaction with the MMP23 pro-domain. In support of this hypothesis, human malignant melanoma tumors with higher MMP23 expression contain significantly fewer tumor-infiltrating lymphocytes, and are associated with a greater risk of recurrence among patients treated with immune biologics [159]. However, we cannot exclude a role for Kv1.3 in the endoplasmic reticulum of tumors.

Overview and future directions

MMP23 is a unique member of the matrix metalloprotease family of proteins with a distinctive domain architecture and function. Moreover, MMP23 appears to be involved in the regulation of Kv1 channel trafficking.

Kv1 channels are abnormally expressed in cancer cells and have been identified as potential targets for the treatment of various cancers [160–164] and autoimmune diseases [165]. Inhibition of Kv1 channels has been shown to induce Bax/Bak-independent death of cancer cells [166] and down-regulate the activity of disease-associated T cells in various autoimmune diseases [167]. Kv channel activity is regulated by mechanisms that control the flow of ions through the pore as well as the trafficking of channels to the cell surface. Dysfunction of Kv channel trafficking can have detrimental consequences. For example, Kv channels play a fundamental role in determining intrinsic neuronal excitability, and channel trafficking defects can lead to aberrant intrinsic excitability and seizures (such as in epilepsy) [168].

MMP23 has been shown to play a role in regulating Kv1 channel activity (Fig. 11) [38, 39]. The MMP23 pro-domain can regulate Kv1 channel trafficking by trapping them in the ER, although it is not known whether this requires a direct interaction or occurs through other unknown mediators. Moreover, the toxin-like domain of MMP23 can block the Kv1.3 pore, obstructing the passage of potassium ions through the channel, although it remains to be determined whether this occurs intracellularly or following cleavage and secretion of the active enzyme.

Fig. 11.

Possible role of various MMP23 domains in modulating Kv channel activity. a Proposed model of MMP23 and Kv1 channels in the ER. The X-ray crystal structure for Kv1.2 (PDB ID: 3LUT) was used. The Kv1 VSD is colored green and the pore domain is orange. The large flexible S1-S2 loops are shown extending out from the Kv1.2 channel. Importantly, the topology of Kv1.2, particularly in the external vestibule region where toxins bind, is similar to that of other Kv1 channels. Black spheres depict potassium ions within the conduction pore. The TMD of the MMP23 pro-domain (gray) transverses the ER membrane while the juxtamembrane helix (α2) is located in the membrane facing the ER lumen. The metalloprotease domain (blue) is represented by the MMP8 catalytic domain structure. Zinc and calcium ions are depicted by magenta and green spheres, respectively. The MMP23 TxD (brown) is situated directly above the outer vestibule of the Kv1 channel. The IgCAM domain is represented by a purple oval. The position of the furin cleavage motif is indicated by a pair of scissors. Adapted from Rangaraju et al. [39]. b Possible modes of MMP23-mediated regulation of Kv1 channel activity. Modulation of Kv1 channel activity may occur by one or more of following mechanisms either individually or in combination. Domains are colored according to (a) while the catalytic zinc is represented by a magenta sphere. (1) The MMP23 pro-domain regulates intracellular trafficking of Kv1 channels by trapping them in the ER. (2) The MMP23 TxD blocks the Kv1 channel pore, attenuating channel activity. (3) Cleavage of loops on the exposed surface of Kv1 channels may lead to modulation of channel activity. It is possible that channel modulation may also occur indirectly by MMP23-mediated proteolytic activation of another protein which then indirectly modulates Kv1 channel activity (not shown). It is possible that interaction of the MMP23 pro-domain with the Kv1 channel may also be required for (2) and (3)

MMPs degrade a wide range of substrates, including proteins of the ECM, signalling molecules and cell-surface receptors [2, 3, 169, 170]. Although Kv channels have not been identified previously as a substrate for MMPs, it is possible that MMP23 may regulate Kv1 activity either indirectly by cleavage of an upstream regulator of channel activity, e.g., serine protease cleavage of protease-activated receptor 2 (PAR₂) leads to the phosphorylation and activation of transient receptor potential (TRP) ion channels [98, 124], while MMP1 and MMP13 have been shown to activate the closely-related protein PAR₁ [171]) or directly by cleavage of the channel itself (e.g., serine protease cleavage of epithelial sodium channels) (Fig. 11b) [132, 133, 172].

The IgCAM domain of MMP23 shares homology with Ig-like C2-type domains of the immunoglobulin superfamily, which mediate protein–protein or protein–lipid interactions [88, 89, 91]. The MMP23 IgCAM domain may act in a manner similar to the Ig domain of Na_Vβ1 subunits by mediating interactions with other proteins or interacting with and modulating the activity of the Kv1.3 channel [137, 138]. Further work is warranted to determine whether the MMP23 IgCAM domain targets substrate proteins analogous to the hemopexin domain of other MMPs or plays a role in modulating intracellular Kv1 trafficking or activity.

MMP23 is widely expressed and is involved in a diverse range of physiological processes [139, 147]. Aberrant MMP23 expression has been found in a number of human cancers [142, 150–152], and we have recently demonstrated the co-localization of MMP23 and Kv1.3 in colon cancer cells [38]. Importantly, Kv1.3 channels have been identified recently as a therapeutic target for the treatment of human cancers [163, 164, 166].

Further studies are now required to (1) identify in vivo binding partners and proteolytic substrates of MMP23, (2) clarify whether the MMP23 pro-domain and MMP23-TxD act independently or cooperatively in binding to Kv1 channels, (3) determine the possible involvement of the IgCAM domain in substrate processing or Kv1 channel modulation, (4) identify MMP23 binding partners that regulate enzyme activity or Kv1 channel trafficking, and (5) define the role (if any) of the N-terminal cytoplasmic tail. Kv1 channels play an essential cellular role and their dysregulation has been implicated in a number of devastating diseases. Characterization of the function and mechanism of MMP23, a unique member of the MMP family, should yield significant insights into physiology and disease.