2.3. Lysyl hydroxylases
Lysyl hydroxylase (EC 1.14.11.4, LH) catalyzes the hydroxylation of lysyl residues in collagens and some other proteins with collagenous domains (Kivirikko & Myllylä 1980, Kivirikko & Myllylä 1984, Kivirikko et al. 1992, Kielty et al. 1993).
At an early stage it was suggested that the same enzyme, protocollagen hydroxylase, catalyzed the hydroxylations of proline and lysine in collagens since they require the same substrates and cofactors (Hausmann 1967, Kivirikko & Prockop 1967, Kivirikko et al. 1968). It was found out later that both enzymatic activities were located at least in different sites in the enzyme (Weinstein et al. 1969). Lysyl hydroxylase was first partially purified from chick embryos and was demonstrated to be a separate enzyme from prolyl hydroxylase by DEAE cellulose chromatography (Kivirikko & Prockop 1972, Popenoe & Aronson 1972). The enzyme was further purified up to several hundred- or thousand-fold from chick embryos and chick embryo cartilage (Ryhänen 1976). By using a procedure involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose, affinity chromatography on a collagen-agarose column and gel filtration (Turpeenniemi et al. 1977), lysyl hydroxylase was finally purified as a homogeneous protein from chick embryos (Turpeenniemi-Hujanen et al. 1980) and from human fetal and placenta tissues (Turpeenniemi-Hujanen et al. 1981).
2.3.1. Molecular properties of lysyl hydroxylase
The active lysyl hydroxylase is a dimer with a molecular weight of about 190 kDa by gel filtration, which consists of only one type of monomer with a molecular weight of about 85 kDa seen in SDS-PAGE (Turpeenniemi-Hujanen et al. 1980, Turpeenniemi-Hujanen et al. 1981, Myllylä et al. 1988). It is a glycoprotein containing asparagine-linked carbohydrate units that are essential for maximal enzyme activity (Myllylä et al. 1988, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998).
Lysyl hydroxylase was first cloned from chick embryos (Myllylä et al. 1991). The mRNA is 4.0 kb in size and the cDNA encodes a polypeptide of 710 amino acid residues with a signal peptide of 20 amino acids at the N-terminus. Surprisingly there is no significant homology in the primary structures between lysyl hydroxylase and the two types of subunit of prolyl 4-hydroxylase, despite the fact that both enzymes share much similarity in their catalytic properties (Myllylä et al. 1991). Molecular cloning and nucleotide sequencing have been reported later for the human (Hautala et al. 1992, Yeowell et al. 1992) and rat lysyl hydroxylases (Armstrong & Last 1995). The mRNA of human lysyl hydroxylase is 3.2 kb. Its subunit consists of 709 amino acid residues and a signal peptide of 18 amino acids. The overall identity between the sequences of human and chick lysyl hydroxylase is 76% at the amino acid level. The carboxytermini are very well conserved between the two enzymes. The human sequence contains 10 cysteine residues, 9 of which are conserved in the chick sequence. The gene for human lysyl hydroxylase (PLOD) is mapped to chromosome 1p36.2-36.3 (Hautala et al. 1992). The mRNA of rat lysyl hydroxylase is 3.2 kb in size. It encodes a protein consisting of 728 amino acid residues, which might contain a signal peptide of 18 amino acids. The complete cDNA of rat lysyl hydroxylase is 91% and 77% identical to those of human and chick at the amino acid level (Armstrong & Last 1995). The chick, human and rat lysyl hydroxylase all contain four potential attachment sites for asparagine-linked oligosaccharides (Myllylä et al. 1991, Hautala et al. 1992, Armstrong & Last 1995).
2.3.2. Reaction catalyzed by lysyl hydroxylase
Lysyl hydroxylase belongs to the group of 2-oxoglutarate dioxygenases. It requires Fe2+, 2-oxoglutarate, O2 and ascorbate in the reaction, and decarboxylates 2-oxoglutarate, one atom of the O2 molecule being incorporated into the succinate while the other is incorporated into the hydroxyl group of the substrate (Kivirikko & Myllylä 1984, Kivirikko et al. 1992).
Figure 2. Hydroxylation reaction catalyzed by lysyl hydroxylase. One atom of oxygen is incorporated into the hydroxyl group of the lysyl residue in the peptide substrate and another one into 2-oxoglutarate, which is decarboxylated to form succinate and liberates CO2.
The hydroxylation reaction involves an ordered binding of Fe2+, 2-oxoglutarate, O2 and the peptide substrate to the enzyme, and an ordered release of the hydroxylated peptide, CO2, succinate, and Fe2+, in which Fe2+ may not necessarily leave the enzyme during each catalytic cycle (Puistola et al. 1980a, 1980b). Lysyl hydroxylase is also able to catalyze the uncoupled decarboxylation of 2-oxoglutarate in the presence of the same cofactors but in absence of the peptide substrate. The reaction rate, however, is only 4% of the one observed in the presence of a saturating concentration of the peptide substrate (Puistola et al. 1980a, 1980b, Myllylä et al. 1984).
2.3.2.1. Peptide substrates
The minimum sequence requirement for lysyl hydroxylase is fulfilled by the -X-Lys-Gly- triplet, but the enzyme purified from chick embryos can also hydroxylate arginine-rich histone, which does not contain any -X-Lys-Gly- triplet but the sequence such as -X-Lys-Ser-, -X-Lys-Ala-, and -X-Lys-Thr- (Ryhänen 1975). This agrees with the presence of the sequences -X-Hyl-Ser- and -X-Hyl-Ala- in the short noncollagenous domains at the end of the α-chains in some collagens (Kivirikko & Myllylä 1980, Kivirikko et al. 1992). It is not yet known whether one single enzyme hydroxylates both -X-Lys-Gly- and other triplets or whether these reactions are catalyzed by different LH isoenzymes in vivo (Ryhänen 1976, Royce & Barnes 1985, Bank et al. 1999).
The interaction with lysyl hydroxylase is also influenced by the amino acid sequence around the lysyl residue, the peptide chain length and the peptide conformation. Lysyl hydroxylase does not act on a -Lys-Gly-Pro- tripeptide whereas -Ile-Lys-Gly- can be hydroxylated. The peptide chain length appears to influence only the Km, which decreases with the increasing chain length, whereas the V of the reaction seems to be unaffected (Kivirikko & Myllylä 1980, Kivirikko et al. 1992). The triple-helical conformation of the substrates completely prevents lysine hydroxylation (Kivirikko & Myllylä 1980, Kivirikko et al. 1992). An extended polyproline-II conformation in the peptide substrate may interact at the binding site on lysyl hydroxylase, while a ‘bent’ structure such as the γ - or β -turn in the -Lys-Gly- segment may be necessary for hydroxylation at the catalytic site (Jiang & Ananthanarayanan 1991, Ananthanarayanan et al. 1992).
2.3.2.2. Cofactors
The hydroxylation reaction catalyzed by lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate as essential cofactors in an ordered mechanism that leads to the release of a hydroxylated lysyl residue in the procollagen polypeptide, CO2, and succinate (Kivirikko & Myllylä 1980, Puistola et al. 1980b, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998). The Km values of the cofactors for lysyl hydroxylase are: 2 µM for Fe2+, 100 µM for 2-oxoglutarate, 45 µM for O2 and 350 µM for ascorbate (Turpeenniemi-Hujanen et al. 1981, Pirskanen et al.1996, Kivirikko & Pihlajaniemi 1998). The Km values for Fe2+, O2 and ascorbate in the case of lysyl hydroxylase is essentially the same as that of prolyl 4-hydroxylase whereas that for 2-oxoglutarate is five times higher than prolyl 4-hydroxylase (Kivirikko & Pihlajaniemi 1998).
The Fe2+ is loosely bound to the enzyme by three side chains (Kivirikko et al. 1992). It doesn’t have to leave the enzyme after every catalytic cycle (Puistola et al. 1980b). Sequence alignment analysis of several 2-oxoglutarate dioxygenases and a related enzyme, isopenicillin N synthase, demonstrates a weak homology within two histidine-containing motifs, His-1 and His-2, located about 50-70 amino acids apart being the residue 656 and 708 in human lysyl hydroxylase. Aspartate residue 658 is also conserved in all enzymes compared (Myllylä et al. 1992). Site-directed mutagenesis studies show that mutations of the conserved His656 to serine, Asp658 to alanine in His-1, and His708 to serine in His-2 completely inactivates human lysyl hydroxylase (Pirskanen et al. 1996), suggesting that the three Fe2+ binding ligands in human lysyl hydroxylase are His656, Asp658, and His708 (Kivirikko & Pihlajaniemi 1998).
2-oxoglutarate is a very specific requirement for the hydroxylation reaction (Kivirikko & Myllylä 1980, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998). However, it can be replaced by 2-oxoadipinate although the Km value is significantly higher for the latter (4.8 mM) (Majamaa et al. 1985). The Km value of 2-oxoglutarate for lysyl hydroxylase is much higher than that of other collagen hydroxylases, implying differences in the structures of the 2-oxoglutarate binding sites, which consist of three distinct subsites. Subsite I is supposed to be a positively charged side chain on the enzyme which binds the C5 carboxyl group of the 2-oxoglutarate. Subsite II is assumed to comprise 2 cis-positioned coordination sites of the enzyme-bound Fe2+, which is chelated by the C1-C2 moiety. Subsite III might involve a hydrophobic binding site in the C3-C4 region of the cofactor (Kivirikko & Pihlajaniemi 1998). Site-directed mutagenesis studies suggest that the residue forming subsite I in human lysyl hydroxylase is Arg718 , which is also located in the His-2 motif of the enzyme and binds the C5 carboxyl group of 2-oxoglutarate (Passoja et al. 1998a).
The molecular oxygen needed for the hydroxylation reaction comes from the atmosphere. The oxygen atoms present in an enzyme-bound intermediate can be exchanged with water (Kikuchi et al. 1983). The first activated intermediate is a dioxygen unit bound to the Fe2+ of the enzyme whereas the final active intermediate carrying out the hydroxylation reaction is probably a ferryl ion (Kivirikko et al. 1992).
Lysyl hydroxylase can complete many catalytic cycles at a maximal rate in the complete absence of ascorbate. Hydroxylation then stops very quickly, however, and ascorbate is required to reactivate the enzyme (Puistola et al. 1980a, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998). Occasionally lysyl hydroxylase catalyzes uncoupled reactions even in the presence of the peptide substrate. In these reactions the reactive iron-oxo complex is probably converted to Fe3+.O-, making the enzyme unavailable for new catalytic cycles until reduced by ascorbate. It is very possible that the main biological function of ascorbate in vivo is to be an alternative oxygen acceptor after such uncoupled cycles (Myllylä et al. 1984). Ascorbate can be replaced by cysteine or dithiothreitol to a minor extent (Puistola et al. 1980a). The studies with prolyl 4-hydroxylase reveal that the ascorbate binding site of the hydroxylation enzyme is partially identical to the binding site of 2-oxoglutarate, and modifications of the ring atoms of ascorbate that abolish the capacity to bind iron destroy the cofactor activity, as in L-galactono γ -lactone and 3-methoxy-L-ascorbate (Majamaa et al. 1986, Kivirikko et al. 1992, Kivirikko & Pihlajaniemi 1998).
Dithiothreitol, bovine serum albumin, and catalase are also needed for lysyl hydroxylase in order to obtain maximal activity in vitro (Kivirikko & Myllylä 1980). The stimulatory effect by dithiothreitol suggests that the catalytic site contains free thiol groups essential for the enzyme activity. The action of bovine serum albumin is partly due to the nonspecific ‘protein effect’, but more likely it is because of the presence of many free thiol groups on this protein. Catalase is probably to act partly by destroying peroxide generated non-enzymatically from Fe2+, O2 and ascorbate, and partly by a nonspecific protein effect as well (Kivirikko & Myllylä 1980, Kivirikko et al. 1992).
2.3.3. Lysyl hydroxylase isoforms
Many findings suggest that lysyl hydroxylase may have tissue-specific or collagen type-specific isoenzymes (Ryhänen 1975, Risteli et al. 1980, Turpeenniemi-Hujanen 1981, Puistola 1982, Puistola & Anttinen 1982, Tajima et al. 1983, Ihme et al. 1984, Royce & Barnes 1985). During the past few years, two novel isoenzymes, termed lysyl hydroxylase 2 (LH2) and lysyl hydroxylase 3 (LH3), have been characterized from human (Valtavaara et al. 1997, Passoja et al. 1998b, Valtavaara et al. 1998) and mouse (Ruotsalainen et al. 1999) tissues. The previous known main isoform is now correspondingly termed lysyl hydroxylase 1 (LH1). In addition, LH2 occurs in two alternatively spliced forms, LH2a and LH2b, in which LH2b contains an additional exon of 63 bp to the LH2a sequence (Valtavaara 1999, Yeowell & Walker 1999a).
The mRNA of human LH2 is 4.2 kb in size. LH2a cDNA encodes a protein of 737 amino acids that includes a signal peptide of 25 residues at the N-terminus whereas LH2b cDNA encodes a protein of 758 amino acids, of which 21 amino acids are encoded by the additional exon 13A (Valtavaara et al. 1997, Valtavaara 1999, Yeowell & Walker 1999a, Yeowell 2002). The overall similarity of amino acid sequence between LH1 and LH2 is over 75%, being even higher at the C-terminus of the molecules. LH2 has ten cysteine residues that are all conserved in LH1, and seven potential N-glycosylation sites (Valtavaara et al. 1997, Yeowell & Walker 1999a). The corresponding gene (PLOD2) has been localized to chromosome 3q23-24 (Szpirer et al. 1997).
The mRNA of human LH3 is 2.8 kb, smaller than that of LH1 and LH2. The cDNA encodes a polypeptide of 738 amino acids including a signal peptide of 24 residues at the N-terminus (Passoja et al. 1998b, Valtavaara et al. 1998, Yeowell 2002). The amino acid sequence shows high identity to LH1 and LH2, both being 59%. The C-terminus is highly conserved in all isoforms. Nine out of ten cysteine residues conserved between LH1 and LH2 are also conserved in LH3. There are only two potential N-glycosylation sites in the molecule. The gene (PLOD3) is localized to chromosome 7q22 (Valtavaara et al. 1998, Valtavaara et al. 2000).
The mRNAs of mouse LH1, LH2, LH3 are 3.0 kb, 3.9 kb, and 3.1 kb, respectively. The cDNA for mouse LH1 encodes a 728 amino acid polypeptide including a putative 18 amino acid N-terminal signal peptide. The mouse LH2 cDNA encodes a protein of 737 amino acids including a putative signal peptide of 27 amino acids. The cDNA of mouse LH3 encodes a polypeptide of 741 amino acids with the first 27 amino acids forming a signal peptide at the N-terminus. The amino acid sequences are approximately 60% identical between the mouse LH isoforms and 91% identical to the corresponding human enzymes (Ruotsalainen et al. 1999). The genes encoding mouse LH isoforms (Plod1, Plod2, Plod3) map to chromosome 4, 5, and 9, respectively (Sipilä et al. 2000).
The complete gene structures for human LH1 (Heikkinen et al. 1994) and LH3 (Rautavuoma et al. 2000), and for mouse LH2 and LH3 (Ruotsalainen et al. 2001) have been characterized so far. Although they basically all have 19 exons and 18 introns, the LH3 genes (Plod3 and PLOD3) are much smaller than PLOD1 and Plod2 due to the existence of many shorter introns in the sequences. Plod2 is the largest gene with an extra exon 13A being alternatively spliced in the processing of mRNA. The sizes of exon 1 and exon 19, which cover most of the 5’ and 3’ ends of the coding regions and the untranslated regions of mRNAs, are very different among these LH isoforms whereas the sizes of the other exons are very similar to each other. The introns of Plod3/PLOD3 constitute only 69-76% of the gene sequence whereas PLOD1 and Plod2 are about 91-92% (Ruotsalainen et al. 2001).
2.3.4. Subcellular localization and distribution of LH isoforms in tissues
LH1 has been demonstrated by a variety of techniques to reside within the lumen of the ER. It appears to be a luminally oriented peripheral membrane protein that associates with the membrane by weak electrostatic interactions (Kivirikko et al. 1992, Kellokumpu et al. 1994, Kivirikko & Pihlajaniemi 1998). Interestingly LH1, as a resident of the ER, does not contain either of the two previously characterized ER-specific retention signals (KDEL or the double lysine motif) in its primary structure (Myllylä et al. 1991, Hautala et al. 1992). It is shown later that cathepsin D, a soluble lysosomal protease, is able to be converted into a membrane-associated protein by tagging a 40-amino acid C-terminal peptide segment of LH1 into the molecule. The first 25 amino acids seem to play a crucial role in terms of membrane association and ER localization. The data thus reveals a novel retrieval mechanism by which the ER lumen can retain its specific protein components from the bulk flow (Suokas et al. 2000). No data is available thus far concerning the subcellular localization of LH2 and LH3.
The human LH1 gene is expressed constitutively in many tissues such as placenta, skin fibroblasts, aorta, lung, vein, artery, cartilage, spleen, gall bladder, brain, liver and skeletal muscle (Heikkinen et al. 1994, Yeowell et al. 1994). Human LH2a is mainly expressed in placenta, pancreas, heart, liver, brain, kidney, skeletal muscle and spleen (Valtavaara et al. 1997, Yeowell & Walker 1999a) whereas LH2b is widely expressed at variable levels in different tissues (Valtavaara 1999, Yeowell & Walker 1999a). LH3 is strongly expressed in heart, placenta and pancreas (Valtavaara et al. 1998). The expression of mouse LH1 is extremely high in liver and heart, as well as in skeletal muscle, kidney and lung (Ruotsalainen et al. 1999). The mouse LH2a and LH2b genes are highly expressed in heart and kidney (Ruotsalainen et al. 1999, Valtavaara 1999) whereas LH3 expression is higher in heart, liver, lung and testis (Ruotsalainen et al. 1999).
The phylogenetic analysis of nine LH sequences from five species suggests that the LH isoforms are derived from one ancestral gene by two duplication events. LH1 and LH2 result from more recent duplications and are more closely related to each other whereas LH3 appears to be the ancestral gene and resembles the C. elegans LH more than the other isoforms (Ruotsalainen et al. 1999).
2.3.5. Ehlers-Danlos syndrome type VI
The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of disorders characterized clinically by joint hypermobility, skin fragility and hyperextensibility and other signs of connective tissue involvement. At least ten different subtypes of EDS have been classified based on genetic, biochemical, and clinical characteristics. The type VI variant of EDS is a recessively inherited connective-tissue disorder with specific features such as muscular hypotonia, kyphoscoliosis, and ocular manifestations (Krane 1984, Steinmann et al. 1993, Yeowell & Pinnell 1993, Byers 1994, Kivirikko & Pihlajaniemi 1998). Patients with EDS VI are biochemically divided into two subclasses: those with a low LH1 activity classified as EDS VIA (Krane et al. 1972, Pinnell et al. 1972, Sussman et al. 1974, Steinmann et al. 1975, Chamson et al. 1987, Wenstrup et al. 1989), and those with normal LH1 activity classified as EDS VIB (Judisch et al. 1976, Ihme et al. 1983, Royce et al. 1989, Steinmann et al. 1993).
Molecular cloning of the human LH1 has made it possible to characterize mutations responsible for this disorder in detail (Yeowell & Walker 2000). The first mutation leading to a deficiency in LH1 activity in EDS VI patients was reported by Hyland and his coworkers (Hyland et al. 1992). Two patients in a family had a homozygous single base substitution in the LH1 gene that converted the CGA codon for Arg319 to a translation termination codon TGA (R319X). The parents and three healthy siblings of the patients were found to be heterozygous carriers of the same mutation (Hyland et al. 1992). The most common mutation seems to be a large seven exon duplication resulting from recombination of Alu-sequences in the LH1 gene. This has been found in approximately 19% of 35 EDS VI families studied (Hautala et al. 1993, Pousi et al. 1994, Heikkinen et al. 1997). Three other mutations have been shown to occur in more than one unrelated EDS VI patient. These include the Y511X mutation in exon 14 (Yeowell & Walker 1997, Walker et al. 1999, Yeowell & Walker 1999b, Pousi et al. 2000), the 15-bp deletion in exon 11 (Yeowell et al. 2000a), and the Q327X mutation in exon 10 (Yeowell et al. 2000b). Other mutations identified so far include Y142X, R670X, the insertion of a single C nucleotide in exon 16 (Yeowell et al. 2000b), a homozygous splice-site mutation that induces the skipping of exon 9 (Pajunen et al. 1998), and some compound heterozygous mutations, for example, a point mutation (exon 19) and a triplet deletion (exon 15) in the two alleles (Ha et al. 1994), a deletion of exon 17 in one allele and a splicing defect resulting in the skipping of exon 16 in the other allele (Pousi et al. 1998), a point mutation in exon 17 of one allele and an unidentified mutation in the other allele (Brinckmann et al. 1998), a nucleotide deletion in the acceptor splice site of intron 4 in one allele, and an insertion of a C nucleotide in exon 2 of the other allele (Heikkinen et al. 1999).