The biosynthesis of collagen is a multistep process. Collagen propeptides are cotranslationally translocated into the ER lumen. Propeptides undergo a number of post-translational modifications. Proline and lysine residues may be hydroxylated by prolyl 3-, prolyl 4- and lysyl hydroxylases. 4-hydroxyproline is essential for intramolecular hydrogen bonding and stability of the triple helical collagenous domain. In fibril forming collagens approximately 50% of prolines are 4-hydroxylated; the extent of this and of 3-hydroxyproline and lysine hydroxylation varies between tissues and collagen types (Kivirikko et al. 1972, 1992). Hydroxylysine molecules can form cross-links between collagen molecules in fibrils, and are sites for glycosyl- and galactosylation. Collagen peptides all have non-collagenous domains; collagens within the subclasses have common chain structures. These non-collagenous domains have regulatory functions; some are biologically active when cleaved from the main peptide chain. Fibrillar collagens all have a large triple helical domain (COL1) bordered by N and C terminal extensions, called the N and C propeptides, which are cleaved prior to formation of the collagen fibril. The C propeptide, also called the NC1 domain, is highly conserved. It directs chain association during intracellular assembly of the procollagen molecule from three collagen propeptide alpha chains (Hulmes 2002). The N-propeptide has a short linker (NC2) connecting the main triple helix to a short minor one (COL2) and a globular N-terminal region NC3. NC3 domains are variable both in size and the domains they contain.
Collagen propeptides typically undergo a number of post-translational modifications. Proline and lysine residues are hydroxylated by prolyl 3-, prolyl 4- and lysyl hydroxylases. 4-hydroxyproline is essential for intramolecular hydrogen bonding and stability of the triple helical collagenous domain. Prolyl 4-hydroxylase may also have a role in alpha chain association as no association of the C-propeptides of type XII collagen was seen in the presence of prolyl 4-hydroxylase inhibitors (Mazzorana et al. 1993, 1996). In fibril forming collagens approximately 50% of prolines are 4-hydroxylated; the extent of this is species dependent, lower hydroxylation correlating with lower ambient temperature and thermal stability (Cohen-Solal et al. 1986, Notbohm et al. 1992). Similarly the extent of 3-hydroxyproline and lysine hydroxylation varies between tissues and collagen types (Kivirikko et al. 1992). Hydroxylysine molecules can form cross-links between collagen molecules in fibrils, and are sites for glycosyl- and galactosylation.
Collagen molecules fold and assemble through a series of distinct intermediates (Bulleid 1996). Individual collagen polypeptide chains are translocated co-translationally across the membrane of the endoplasmic reticulum (ER). Intra-chain disulfide bonds are formed within the N-propeptide, and hydroxylation of proline and lysine residues occurs within the triple helical domain (Kivirikko et al. 1992). When the peptide chain is fully translocated into the ER lumen the C-propeptide folds, the conformation being stabilized by intra-chain disulfide bonds (Doege and Fessler 1986). Pro alpha-chains associate via the C-propeptides (Byers et al. 1975, Bachinger et al. 1978), or NC2 domains for FACIT family collagens (Boudko et al. 2008) to form an initial trimer which can be stabilized by the formation of inter-chain disulfide bonds (Schofield et al. 1974, Olsen et al. 1976), though these are not a prerequisite for further folding (Bulleid et al. 1996). The triple helix then nucleates and folds in a C- to N- direction. The association of the individual chains and subsequent triple helix formation are distinct steps (Bachinger et al. 1980). The N-propeptides associate and in some cases form inter-chain disulfide bonds (Bruckner et al., 1978). Procollagen is released via carriers into the exracellular space (Canty & Kadler 2005). Fibrillar procollagens undergo removal of the C- and N-propeptides by procollagen C and N proteinases respectively, both Zn2+ dependent metalloproteinases. Propeptide processing is a required step for normal collagen I and III fibril formation, but collagens can retain some or all of their non-collagenous propeptides. Retained collagen type V and XI N-propeptides contribute to the control of fibril growth by sterically limiting lateral molecule addition (Fichard et al. 1995). Processed fibrillar procollagen is termed tropocollagen, which is considered to be the unit of higher order fibrils and fibres. Tropocollagens of the fibril forming collagens I, II, III, V and XI sponteneously aggregate in vitro in a manner that has been compared with crystallization, commencing with a nucleation event followed by subsequent organized aggregation (Silver et al. 1992, Prockop & Fertala 1998). Fibril formation is stabilized by lysyl oxidase catalyzed crosslinks between adjacent molecules (Siegel & Fu 1976).
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The C-propeptides of collagen propeptide chains are essential for the association of three peptide chains into a trimeric but non-helical procollagen. This initial binding event determines the composition of the trimer, brings the individual chains into the correct register and initiates formation of the triple helix at the C-terminus, which then proceeds towards the N-terminus in a zipper-like fashion (Engel & Prockop 1991). Most early refolding studies were performed with collagen type III, which contains a disulfide linkage at the C-terminus of its triple helix (Bächinger et al. 1978, Bruckner et al. 1978) that acts as a permanent linker even after removal of the non-collagenous domains.
Mutations within the C-propeptides further suggest that they are crucial for the correct interaction of the three polypeptide chains and for subsequent correct folding (refs. in Boudko et al. 2011).
COL25A1 is a brain-specific membrane-bound collagen. Proteolytic processing releases CLAC (collagenous Alzheimer amyloid plaque component), a soluble form of COL25A1 containing the extracellular collagen domains. CLAC associates with amyloid beta peptides (Abeta) in the brains of patients with Alzheimer's disease, inhibiting amyloid fibril formation (Osada et al. 2005).
Collagen type VI forms dimers and tetramers before secretion (Furthmayr et al. 1983, von der Mark et al. 1984, Engel et al. 1985, Colombatti et al. 1987). The monomers associate in antiparallel with a 30nm axial shift, intertwining 4 or 5 times (Furthmayr et al. 1983). These associate laterally to form tetramers (Furthmayr et al. 1983, von der Mark et al. 1984)The tetramers associate to form microfibrils in a non-covalent manner, presumed to be mediated through A domain interactions (Baldock et al. 2003). Collagen type VI chains are extensively post translationally modifed by the hydroxylation of proline and lysine residues (Myllyharju & Kivirikko 2004) and subsequent glycosylation of hydroxylysine, thought to be essential for tetramer formation and secretion (Sipila et al. 2007).
Collagen VI dimers combine to form tetramers before secretion (Furthmayr et al. 1983, von der Mark et al. 1984, Engel et al. 1985, Engvall et al. 1986, Colombatti et al. 1987). Collagen type VI chains are extensively post translationally modifed by the hydroxylation of proline and lysine residues (Myllyharju & Kivirikko 2004) and subsequent glycosylation of hydroxylysine, thought to be essential for tetramer formation and secretion (Sipila et al. 2007).
Collagen was for many years considered the only source of 4-hydroxyproline (4-Hyp) in animals. Though it is now known that other proteins such as C1q and elastin also contain 4-Hyp, collagen is by far the major source (Adams & Frank 1980). 4-Hyp is required for collagen stability at physiological temperatures. The abundance of Hyp in animal proteins is ~4%, making it more abundant than the amino-acids Cys, Gln, His, Met, Phe, Trp and Tyr (McCaldon & Argos 1988). In collagen Hyp abundance is much higher at ~38% (Ramshaw et al. 1998). Full collagen proline hydroxylation significantly raises the melting temperature (Tm) by stabilizing the collagen triple helix (Berg & Prockop 1973a), a process that has been studied extensively using synthetic collagen peptides (Sakakibara et al. 1973, Holmgren et al. 1998) and is well understood at the structural level (Shoulders & Raines 2009). Collagen 4-Hyp content is relatively stable, with small differences between collagen types. Collagen type I has approximately 1 4-Hyp for every 10 residues, roughly 50% of available prolines (Kivirikko et al. 1992). Conversion of Pro to (2S,4R)-4-hydroxyproline (4-Hyp) is the most prevalent posttranslational modification in humans, catalyzed by prolyl 4-hydroxylase (P4H). Mammalian prolyl 4-hydroxylase is an alpha2 beta2 tetramer (Berg & Prockop 1973b). The 59-kDa alpha subunit contains the substrate-binding domain and the enzymic active site (Helaakoski et al. 1989). Humans and most other vertebrates have three isoforms of the alpha subunit, isoform alpha-1 is the most prevalent. The pair of alpha subunits can be any of the three isoforms (Gorres & Raines 2010). The 55-kDa beta subunit is protein disulphide isomerase (PDI), which has additional functions in collagen formation. As part of P4H it retains the tetramer in the ER lumen and maintains the otherwise insoluble alpha subunit in an active form (Vuori et al. 1992, Nietfeld & Kemp 1981). P4H is a member of the non-heme iron(II), alpha-ketoglutarate-dependent dioxygenase family. Molecular oxygen (O2), 2-oxoglutarate (alpha-ketoglutarate) and iron(II) are required for its activity (Hutton & Udenfriend 1966). During the reaction, alpha-ketoglutarate is oxidatively decarboxylated producing succinate and CO2 (Rhoads & Udenfriend 1968, Gorres & Raines 2010). Ascorbate is required as a cofactor but not consumed (Kivirikko et al 1989). The minimum substrate required for hydroxylation is an Xaa-Pro-Gly tripeptide, with Pro preferred in the Xaa position, though hydroxylation can occur at lower rates with a variety of residues at this position (Kivirikko et al. 1972). A number of other peptides, notably elastin, are substrates for P4H (Bhatnagar 1978).
For brevity, all forms of collagen propeptide are shown as having 3X 4-Hyp residues following the action of P4H.
Collagen contains (2S,3S)-3-hydroxyproline (3-Hyp), though much less abundantly than 4-Hyp (Rhodes and Miller 1978). The 3-Hyp content of collagen is much more variable than that of 4-Hyp, varying between collagen types, tissues, developmental stages and pathological states (Kivirikko et al. 1992). It is more prevalent in type IV and V collagens at 10-15 3-Hyp residues (Bentz et al. 1983) than in Type I-III fibrillar collagens which have a single 3-Hyp residue per chain; the alpha-1 chain of type I collagen has 3-Hyp at residue 986 (Fietzek et al. 1972, Marini et al. 2007). 3-Hyp is formed from Pro in the Xaa position of Xaa-Hyp-Gly triplets (Gryder et al. 1975, Kivirikko et al. 1992). It is likely that 4-Hyp is a requirement at the second position of the triplet as 4-Hyp rich substrates are more active than 4-Hyp poor (Adams & Frank 1980). 3-Hyp has a modest effect on triple-helix stability (Jenkins et al. 2003; Mizuno et al. 2008). 3-Hyp may adjust the stability of basement membranes to enable formation of the meshwork structure, or serve as a ligand for other proteins. It is suggested to have a role in the self-assembly of collagen supramolecular structures (Weis et al. 2010). 3-Hyp is formed by prolyl 3-hydroxylase (P3H; EC 1.14.11.7), which has 3 isoforms in vertebrates. All contain an ER-retention signal but vary in their tissue expression (Vranka et al. 2009). P3H can hydroxylate prolines that precede 4-Hyp residues (Tryggvason et al. 1976) but not those that precede an unhydroxylated proline (Kivirikko & Myllla 1982, Myllyharju 2005). Like P4H, P3H requires molecular oxygen, alpha-ketoglutarate, iron(II), and ascorbate for activity. P3H1 is homologous to mammalian leprecan or growth suppressor 1 (Gros1), and forms a 3-prolyl hydroxylation complex with cartilage-associated protein (CRTAP) and a peptidyl-prolyl cis-trans isomerase, cyclophilin B (CypB), which is encoded by the PPIB gene (Vranka et al. 2004). Lack of 3-Hyp in Type I and II collagens leads to an osteogenesis imperfecta (OI)-like disease, as demonstrated by CRTAP and PPIB knock-out mice (Morello et al. 2006, Choi et al. 2009) and by mutations in human LEPRE1 (which encodes P3H1), CRTAP, and PPIB (Barnes et al. 2006, Cabral et al. 2007, van Dijk et al. 2009). The P3H1/CRTAP/CypB complex has also been shown to have chaperone activity (Ishikawa et al. 2009). P3H2 hydroxylates peptides derived from Type IV collagen more efficiently than Type I peptides and is localized to tissues that are rich in basement membrane (Tiainen et al. 2008). The effect of prolyl 3-hydroxylation on basement membrane collagens remains unknown.
In this generalized reaction, all collagen subtypes are represented as having one 3-Hyp residue.
Lysyl hydroxylase (LH) (E.C. 1.14.11.4) is a dimeric enzyme that catalyzes the formation of (2S,5R)-5-hydroxylysyl residues (5-Hyl) in proteins (reviewed in Myllyharju & Kivirikko 2001) within a peptide linkage at the Y position of the repeating X-Y-Gly sequence motif. The extent of 5-Hyl formation is much more variable than that of hydroxyproline. It varies between collagen types, tissues and by physiological state (Miller 1984). 5-Hyl content also differs between the helical and telopeptide domains. This and the observation that purified lysyl hydroxylase failed to hydroxylate Lys in the telopeptide domains has led to speculation that there are separate enzymes responsible for Lys hydroxylation in the helical and telopeptide domains (Royce & Barnes 1985, Gerriets et al. 1993). The LH2b isoform may be the telopeptide-specific form (Pornprasertsuk et al. 2004). In human type I collagen, there are 38 residues of Lys in each alpha-1 chain (36 in the helical domain, 1 each in the C- and N-telopeptide domains) and 31 in each alpha-2 chain (30 in the helical domain,1 in the N-telopeptide and none in the C-telopeptide domains) (Yamauchi & Shiiba 2002).
LH requires ferrous iron, oxygen, 2-oxoglutarate, and ascorbate. The hydroxylation reaction occurs during collagen biosynthesis in the ER as a co- and post-translational event, before triple helix formation. Three LH isoforms have been characterized in humans, encoded by the genes PLOD1-3. The isoforms appear to have preferences for certain collagen types, e.g. LH3 preferentially binds collagen types IV, VI, XI and XII (Myllyla et al. 2007). LH3 has galactosyltransferase and glucosyltransferase activities in addition to its lysyl hydroxylase activity (Heikkinen et al. 2000, Wang et al. 2002), a multifunctionality also seen in the single C. elegans orthologue (Wang et al. 2002a, b). Hydroxylysine residues can form stable intermolecular cross-links between collagen molecules in fibrils and also represent sites for glucosyl- and galactosyl- carbohydrate attachment.
In this reaction all collagen subtypes are represented as having a single hydroxylysine.
Hydroxylysine glycosides are specific to collagen. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation. Either galactose or glucose-galactose are attached to approximately one third of hydroxylysine residues by specific transferases, beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase, forming galactosyl hydroxylysine (Gal-Hyl) and glucosyl-galactosyl hydroxylysine (Glu-Gal-Hyl) respectively. The genes GLT25D1 and GLT25D2 encode galactosyltransferases that are active with various types of collagen and the serum mannose-binding lectin MBL, which also contains a collagen domain. GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene was found to be expressed only at low levels in the nervous system. These galactosyltransferases convert 5-hydroxylysine to 5-galactosyl hydroxylysine (Gal-Hyl). The extent of hydroxylysine galactosylation is variable between collagen types and locations; it is particularly common in bone type I collagen (Al-Dehaimi et al. 1999). Although the fraction of hydroxylysine residues that are glycosylated does not differ between skin and bone (the major sources of type I collagen) the pattern of hydroxylysine glycosylation is different. Glu-Gal-Hyl predominates in skin, where the Glu-Gal-Hyl/Gal-Hyl ratio is approximately 2 (Pinnell et al. 1971), whereas Gal-Hyl predominates in bone, where the Glu-Gal-Hyl/Gal-Hyl ratio is 0.47 (Krane et al. 1977).
The ER membrane-associated enzyme PLOD3 has collagen galactosyltransferase activity (Heikkinen et al. 2000, Wang et al. 2002) though the biological significance of this has been questioned (Schegg et al. 2009).
Fibrillar collagen is synthesized in the ER as procollagen with N- and C-terminal propeptides flanking the collagenous domain (Bellamy & Bornstein 1971). These propeptides, particularly the C-propeptide, inhibit fibril formation (Kadler et al. 1987). Type V collagen N-propeptide removal is partial, and reported to be mediated by BMP-1 which cleaves between the proline/arginine-rich protein domain and the variable domain of the alpha1 chain and between the small and the large collagenous domain of alpha3 chain (refs. in Colige et al. 2005).
Mutations in procollagen peptide type V (COL5A1) can cause diseases such as Ehlers-Danlos Sydrome. Majority of the cases result from haploinsufficiency of COL5A1 protein that may lead to a disorganised extracellular matrix. Moreover, other mutations result in structural defects which may in turn result in defective collagen fibrils. The accumulation and interaction of this defective collagen fibrils may lead to ER stress and other complications in the cell (Takahara K et al. 2002, Symoens S et al. 2012).
Fibrillar collagen is synthesized in the ER as procollagen with N- and C- terminal propeptides flanking the collagenous domain (Bellamy & Bornstein 1971). These propeptides, particularly the C-propeptide, inhibit fibril formation (Kadler et al. 1987). Removal of propeptides is generally described as an extracellular process but can occur within the cell. Procollagen processing in tendon fibroblasts was initiated within the secretory pathway with the N-propetides removed first, in the ER or an intermediate between the ER and Golgi. The C peptides were removed later, probably at the cell membrane-ECM interface (Canty-Laird et al. 2012).
Collagen C-propeptides are cleaved by the tolloid family metalloproteinases bone morphogenic protein 1 (BMP1)/mammalian tolloid (mTLD), tolloid-like 1 (TLL1) and TLL2.
Procollagen types I-III are cleaved by BMP1/mTLD (Chicken types I and II, human type III, by chicken enzyme, Hojima et al. 1985, human types I, II, human enzyme, Scott et al. 1999), TLL-1 (human types I, II, human enzyme, Scott et al. 1999), and TLL-2 in the presence of PCOLCE (PCPE-1, Pappano et al. 2003, Petropoulou et al. 2005). Type V C-propeptide removal is mediated by furin-like proprotein convertases and/or BMP-1 depending on the chain type (Kessler et al. 2005).
As the collagen peptide chain is translocated across the membrane of the endoplasmic reticulum, intrachain disulfide bonds are formed within the N- and C-propeptides. This allows the triple helical domain to form a nucleation point at its C-terminal end and ensures correct alignment of the chains (Engel & Prockop 1991). Protein disulfide isomerase (P4HB) catalyzes the formation of both intra- (Bulleid & Freedman 1988) and inter-chain disulfide bonds (Koivu & Myllylä 1987). In addition, PDI acts as a molecular chaperone, interacting with monomeric collagen propeptide chains to prevent premature assembly or aggregation (Wilson et al. 1998).
Alignment of the C-terminal prodomains initiates triple helix formation, which propagates in a zipper-like fashion in the C-to-N direction. This occurs in the rough endoplasmic reticulum (Engel & Prockop 1991). Compared with the folding of globular proteins and coiled-coil structures, the concentration-independent folding steps of collagen are extremely slow (Bächinger et al. 1980). Triple helix formation combines a fast process, interpreted as the folding of regions devoid of cis residues, and a slow process, limited by the slow kinetics of cis to trans prolyl-isomerization (Bächinger et al. 1978). Triple-helix formation in regions devoid of cis-prolyl bonds is 3-4 times faster than formation limited by prolyl isomerization reactions (Bachmann et al. 2005). Conversion to trans is required as only trans-peptide bonds can be incorporated into the collagen triple helix (Zeng et al. 1998). Efficient helix folding requires the presence of the 3-prolyl hydroxylation complex. This trimer of Prolyl 3-hydroxylase 1 (LEPRE1), Cyclophilin B (CyPB), also called Peptidyl-prolyl cis-trans isomerase B (PPIB) and CRTAP has 3-prolyl hydroxylase, PPIase and procollagen chaperone properties (Ishikawa et al. 2009, van Dijk et al. 2009). Efficient folding involves additional collagen-specific chaperones such as Serpin H1 (HSP47 - Smith et al. 1995). CyPB belongs to the cyclophilins, a conserved class of intracellular and/or secreted proteins originally identified as cellular binding proteins for the immunosuppressive drug cyclosporin A. They are peptidyl-prolyl cis-trans isomerases (PPIases), which catalyze the cis-trans isomerisation of peptide bonds. CyPB localises to the rough ER but is also secreted extracellularly. It directly interacts with procollagen and is believed to be responsible for converting procollagen cis- to trans-conformers (Zeng et al. 1998). CyPB and Serpin H1 are also involved in procollagen export and secretion. Results obtained with collagen peptides suggest that variations in the Gly-X-Y sequence are likely to result in a non-uniform helical twist along the length of a collagen fibril. Sequences poor in imino acids will have a symmetry close to 10 tripeptide units for every 3 turns of the triple helix (10/3), while stretches of Gly-Pro-Hyp units may have 7/2 symmetry (Brodsky & Persikov 2005).
Collagen precursors are co-translated into the ER, where post-translational modifications occur that are essential for oligomerization and stabilization, the latter thought to involve Serpin H1 (HSP47). Trimers are exported from the ER and trafficked through the Golgi network before secretion into the extracellular space and organization into higher order structures. The precise route and mechanism of secretion for collagen is unclear (Canty & Kadler 2005). In the conventional protein secretion pathway cargo is collected at ER exits sites and loaded into small membrane vesicles that are generated by a set of highly conserved proteins called the coat protein complex II (COPII) (Jensen & Schekman 2011). However, these small vesicles of 60-90 nm diameter are too small for collagens some of which assemble in the ER into 300-400 nm rod-like structures. Collagen secretion appears to involve a modification of the COPII process involving TANGO1 and cTAGE5 which form a dimer at ER exit sites (Malhotra & Erlmann 2011). TANGO1-null mice die at birth and are defective in the secretion of a number of different collagens (Wilson et al. 2011). Another recent study suggests that ubiquitination of one of the COPII complex proteins, SEC31, is sufficient to allow formation of vesicles that are large enough to hold procollagen (Jin et al. 2012).
Secretion of transmembrane collagens is presumably similar to the secretion of extracellular forms except that they are trafficked to the plasma membrane rather than the extracellular space.
The ER membrane-associated enzyme PLOD3 has collagen galactosyltransferase activity (Heikkinen et al. 2000, Wang et al. 2002), though whether it physiologically relevant has been questioned (Schegg et al. 2009).
The ER membrane-associated enzyme PLOD3 has collagen galactosyltransferase activity (Heikkinen et al. 2000, Wang et al. 2002), though the biological significance of this has been questioned (Schegg et al. 2009).
Hydroxylysine glycosides are only found in collagen. Glycosylation takes place in the endoplasmic reticulum, before triple-helix formation. Either galactose or glucose-galactose are attached to approximately one third of hydroxylysine residues by specific transferases, beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase, forming galactosyl hydroxylysine (Gal-Hyl) and glucosyl-galactosyl hydroxylysine (Glu-Gal-Hyl) respectively. The genes COLGALT1 and COLGALT2 encode galactosyltransferases that are active with various types of collagen and the serum mannose-binding lectin MBL, which also contains a collagen domain. COLGALT1 is constitutively expressed in human tissues, whereas the COLGALT2 was found to be expressed only at low levels in the nervous system. These galactosyltransferases convert 5-hydroxylysine to 5-galactosyl hydroxylysine (Gal-Hyl). The extent of hydroxylysine galactosylation is variable between collagen types and locations; it is particularly common in bone type I collagen (Al-Dehaimi et al. 1999). Although the fraction of hydroxylysine residues that are glycosylated does not differ between skin and bone (the major sources of type I collagen) the pattern of hydroxylysine glycosylation is different. Glu-Gal-Hyl predominates in skin, where the Glu-Gal-Hyl/Gal-Hyl ratio is approximately 2 (Pinnell et al. 1971), whereas Gal-Hyl predominates in bone, where the Glu-Gal-Hyl/Gal-Hyl ratio is 0.47 (Krane et al. 1977).
Collagen contains (2S,3S)-3-hydroxyproline (3-Hyp), though much less abundantly than 4-Hyp (Rhodes and Miller 1978). The 3-Hyp content of collagen is much more variable than that of 4-Hyp, varying between collagen types, tissues, developmental stages and pathological states (Kivirikko et al. 1992). It is more prevalent in type IV and V collagens at 10-15 3-Hyp residues (Bentz et al. 1983) than in Type I-III fibrillar collagens which have a single 3-Hyp residue per chain; the alpha-1 chain of type I collagen has 3-Hyp at residue 986 (Fietzek et al. 1972, Marini et al. 2007). 3-Hyp is formed from Pro in the Xaa position of Xaa-Hyp-Gly triplets (Gryder et al. 1975, Kivirikko et al. 1992). It is likely that 4-Hyp is a requirement at the second position of the triplet as 4-Hyp rich substrates are more active than 4-Hyp poor (Adams & Frank 1980). 3-Hyp has a modest effect on triple-helix stability (Jenkins et al. 2003; Mizuno et al. 2008). 3-Hyp may adjust the stability of basement membranes to enable formation of the meshwork structure, or serve as a ligand for other proteins. It is suggested to have a role in the self-assembly of collagen supramolecular structures (Weis et al. 2010). 3-Hyp is formed by prolyl 3-hydroxylase (P3H; EC 1.14.11.7), which has 3 isoforms in vertebrates. All contain an ER-retention signal but vary in their tissue expression (Vranka et al. 2009). P3H can hydroxylate prolines that precede 4-Hyp residues (Tryggvason et al. 1976) but not those that precede an unhydroxylated proline (Kivirikko & Myllla 1982, Myllyharju 2005). Like P4H, P3H requires molecular oxygen, alpha-ketoglutarate, iron(II), and ascorbate for activity. P3H1 is homologous to mammalian leprecan or growth suppressor 1 (Gros1), and forms a 3-prolyl hydroxylation complex with cartilage-associated protein (CRTAP) and a peptidyl-prolyl cis-trans isomerase, cyclophilin B (CypB), which is encoded by the PPIB gene (Vranka et al. 2004). Lack of 3-Hyp in Type I and II collagens leads to an osteogenesis imperfecta (OI)-like disease, as demonstrated by CRTAP and PPIB knock-out mice (Morello et al. 2006, Choi et al. 2009) and by mutations in human LEPRE1 (which encodes P3H1), CRTAP, and PPIB (Barnes et al. 2006, Cabral et al. 2007, van Dijk et al. 2009). The P3H1/CRTAP/CypB complex has also been shown to have chaperone activity (Ishikawa et al. 2009). P3H2 hydroxylates peptides derived from Type IV collagen more efficiently than Type I peptides and is localized to tissues that are rich in basement membrane (Tiainen et al. 2008). The effect of prolyl 3-hydroxylation on basement membrane collagens remains unknown.
Hydroxylysine glycosides are specific to collagen. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation. Either galactose or glucose-galactose are attached to approximately one third of hydroxylysine residues by specific transferases, beta(1-O)galactosyl- and alpha(1-2)glucosyltransferase, forming galactosyl hydroxylysine (Gal-Hyl) and glucosyl-galactosyl hydroxylysine (Glu-Gal-Hyl) respectively. The genes COLGALT1 and COLGALT2 encode galactosyltransferases that are active with various types of collagen and the serum mannose-binding lectin MBL, which also contains a collagen domain. COLGALT1 is constitutively expressed in human tissues, whereas the COLGALT2 was found to be expressed only at low levels in the nervous system. These galactosyltransferases convert 5-hydroxylysine to 5-galactosyl hydroxylysine (Gal-Hyl). The extent of hydroxylysine galactosylation is variable between collagen types and locations; it is particularly common in bone type I collagen (Al-Dehaimi et al. 1999). Although the fraction of hydroxylysine residues that are glycosylated does not differ between skin and bone (the major sources of type I collagen) the pattern of hydroxylysine glycosylation is different. Glu-Gal-Hyl predominates in skin, where the Glu-Gal-Hyl/Gal-Hyl ratio is approximately 2 (Pinnell et al. 1971), whereas Gal-Hyl predominates in bone, where the Glu-Gal-Hyl/Gal-Hyl ratio is 0.47 (Krane et al. 1977).
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procollagen triple
helices:Serpin H1Mutations within the C-propeptides further suggest that they are crucial for the correct interaction of the three polypeptide chains and for subsequent correct folding (refs. in Boudko et al. 2011).
and
tropocollagens:Serpin H1hydroxylases:Lysyl hydroxylase procollagen
substrateshydroxylases:Lysyl hydroxylated collagen
propeptidescollagen
propeptidesdimer:Lysyl hydroxylated collagen
propeptidesAnnotated Interactions
procollagen triple
helices:Serpin H1and
tropocollagens:Serpin H1hydroxylases:Lysyl hydroxylase procollagen
substrateshydroxylases:Lysyl hydroxylated collagen
propeptideshydroxylases:Lysyl hydroxylated collagen
propeptidescollagen
propeptidescollagen
propeptidescollagen
propeptidesdimer:Lysyl hydroxylated collagen
propeptidesdimer:Lysyl hydroxylated collagen
propeptidesFor brevity, all forms of collagen propeptide are shown as having 3X 4-Hyp residues following the action of P4H.
3-Hyp is formed by prolyl 3-hydroxylase (P3H; EC 1.14.11.7), which has 3 isoforms in vertebrates. All contain an ER-retention signal but vary in their tissue expression (Vranka et al. 2009). P3H can hydroxylate prolines that precede 4-Hyp residues (Tryggvason et al. 1976) but not those that precede an unhydroxylated proline (Kivirikko & Myllla 1982, Myllyharju 2005). Like P4H, P3H requires molecular oxygen, alpha-ketoglutarate, iron(II), and ascorbate for activity. P3H1 is homologous to mammalian leprecan or growth suppressor 1 (Gros1), and forms a 3-prolyl hydroxylation complex with cartilage-associated protein (CRTAP) and a peptidyl-prolyl cis-trans isomerase, cyclophilin B (CypB), which is encoded by the PPIB gene (Vranka et al. 2004). Lack of 3-Hyp in Type I and II collagens leads to an osteogenesis imperfecta (OI)-like disease, as demonstrated by CRTAP and PPIB knock-out mice (Morello et al. 2006, Choi et al. 2009) and by mutations in human LEPRE1 (which encodes P3H1), CRTAP, and PPIB (Barnes et al. 2006, Cabral et al. 2007, van Dijk et al. 2009). The P3H1/CRTAP/CypB complex has also been shown to have chaperone activity (Ishikawa et al. 2009). P3H2 hydroxylates peptides derived from Type IV collagen more efficiently than Type I peptides and is localized to tissues that are rich in basement membrane (Tiainen et al. 2008). The effect of prolyl 3-hydroxylation on basement membrane collagens remains unknown.
In this generalized reaction, all collagen subtypes are represented as having one 3-Hyp residue.
In human type I collagen, there are 38 residues of Lys in each alpha-1 chain (36 in the helical domain, 1 each in the C- and N-telopeptide domains) and 31 in each alpha-2 chain (30 in the helical domain,1 in the N-telopeptide and none in the C-telopeptide domains) (Yamauchi & Shiiba 2002).
LH requires ferrous iron, oxygen, 2-oxoglutarate, and ascorbate. The hydroxylation reaction occurs during collagen biosynthesis in the ER as a co- and post-translational event, before triple helix formation. Three LH isoforms have been characterized in humans, encoded by the genes PLOD1-3. The isoforms appear to have preferences for certain collagen types, e.g. LH3 preferentially binds collagen types IV, VI, XI and XII (Myllyla et al. 2007). LH3 has galactosyltransferase and glucosyltransferase activities in addition to its lysyl hydroxylase activity (Heikkinen et al. 2000, Wang et al. 2002), a multifunctionality also seen in the single C. elegans orthologue (Wang et al. 2002a, b). Hydroxylysine residues can form stable intermolecular cross-links between collagen molecules in fibrils and also represent sites for glucosyl- and galactosyl- carbohydrate attachment.
In this reaction all collagen subtypes are represented as having a single hydroxylysine.
Mutations in procollagen peptide type V (COL5A1) can cause diseases such as Ehlers-Danlos Sydrome. Majority of the cases result from haploinsufficiency of COL5A1 protein that may lead to a disorganised extracellular matrix. Moreover, other mutations result in structural defects which may in turn result in defective collagen fibrils. The accumulation and interaction of this defective collagen fibrils may lead to ER stress and other complications in the cell (Takahara K et al. 2002, Symoens S et al. 2012).Collagen C-propeptides are cleaved by the tolloid family metalloproteinases bone morphogenic protein 1 (BMP1)/mammalian tolloid (mTLD), tolloid-like 1 (TLL1) and TLL2.
Procollagen types I-III are cleaved by BMP1/mTLD (Chicken types I and II, human type III, by chicken enzyme, Hojima et al. 1985, human types I, II, human enzyme, Scott et al. 1999), TLL-1 (human types I, II, human enzyme, Scott et al. 1999), and TLL-2 in the presence of PCOLCE (PCPE-1, Pappano et al. 2003, Petropoulou et al. 2005). Type V C-propeptide removal is mediated by furin-like proprotein convertases and/or BMP-1 depending on the chain type (Kessler et al. 2005).
3-Hyp is formed by prolyl 3-hydroxylase (P3H; EC 1.14.11.7), which has 3 isoforms in vertebrates. All contain an ER-retention signal but vary in their tissue expression (Vranka et al. 2009). P3H can hydroxylate prolines that precede 4-Hyp residues (Tryggvason et al. 1976) but not those that precede an unhydroxylated proline (Kivirikko & Myllla 1982, Myllyharju 2005). Like P4H, P3H requires molecular oxygen, alpha-ketoglutarate, iron(II), and ascorbate for activity. P3H1 is homologous to mammalian leprecan or growth suppressor 1 (Gros1), and forms a 3-prolyl hydroxylation complex with cartilage-associated protein (CRTAP) and a peptidyl-prolyl cis-trans isomerase, cyclophilin B (CypB), which is encoded by the PPIB gene (Vranka et al. 2004). Lack of 3-Hyp in Type I and II collagens leads to an osteogenesis imperfecta (OI)-like disease, as demonstrated by CRTAP and PPIB knock-out mice (Morello et al. 2006, Choi et al. 2009) and by mutations in human LEPRE1 (which encodes P3H1), CRTAP, and PPIB (Barnes et al. 2006, Cabral et al. 2007, van Dijk et al. 2009). The P3H1/CRTAP/CypB complex has also been shown to have chaperone activity (Ishikawa et al. 2009). P3H2 hydroxylates peptides derived from Type IV collagen more efficiently than Type I peptides and is localized to tissues that are rich in basement membrane (Tiainen et al. 2008). The effect of prolyl 3-hydroxylation on basement membrane collagens remains unknown.