71Osteogenesis Imperfecta.DOI:

© 2012 Elsevier Inc. All rights reserved.Portions of the contribution are the public domain

2014http://dx.doi.org/10.1016/B978-0-12-397165-4.00007-1

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INTRODUCTION: COLLAGEN ROLE IN OSTEOGENESIS IMPERFECTA (OI)

Most OI cases are caused by dominantly inherited mutations in COL1A1 and COL1A2 genes, which encode the α1(I) and α2(I) chains of type I collagen.1 Recently discovered mutations in other genes, which cause reces-sively inherited OI, further emphasize the central role of type I collagen in this disorder. Among the latter genes, CRTAP, LEPRE1, PPIB, SERPINH1, FKBP10, PLOD2 and BMP1 encode proteins essential for normal production or function of type I collagen.2–15 SERPINF1 encodes a pro-tein that has high binding affinity to type I collagen and might be involved in regulating type I collagen function, although no defects in type I collagen biosynthesis have been detected in cultures of SERPINF1-deficient dermal fibroblasts from patients with type VI OI.16,17 In fact, the only report of OI without direct links to type I collagen describes a single patient with a frameshift mutation in the SP7 gene, which encodes a transcription factor involved in osteoblast differentiation and function.18

All these recent advances in OI genetics underscore the long-held belief that understanding regulation of type I collagen biosynthesis, structure and function is crucial for understanding the underlying causes and treatment of OI. Several other chapters in this book discuss protein machin-ery involved in collagen biosynthesis, including the protein deficiencies which cause the newly discovered recessive forms of OI. In the present chapter, we focus on type I col-lagen properties that appear to be important in OI.

TYPE I COLLAGEN

Type I collagen is by far the most abundant protein in all vertebrates. It assembles into fibers that form the structural and mechanical scaffold (matrix) of bone, skin, tendons, cornea, blood vessel walls and other con-nective tissues. Heterotrimers of two α1(I) and one α2(I) chains are the dominant isoform of type I collagen. Homotrimers of three α1(I) chains are found in fetal tis-sues,19 tumors20–25 and some fibrotic lesions.26–28 The homotrimeric isoform is more resistant to cleavage by collagenases, which might explain its accumulation and functional role in tumors and fibrotic lesions.25,29 However, this isoform becomes prevalent in other tissues only in extremely rare disorders associated with COL1A2 deficiency.30–32

Type I collagen is synthesized as a procollagen pre-cursor, which consists of an N-terminal propeptide, central collagen domain and C-terminal propeptide. Procollagen chains are cotranslationally translocated into the lumen of rough endoplasmic reticulum (ER). After post-translational modification and fold-ing in the ER, procollagen molecules are transported through Golgi and secreted from the cell. N- and C-propeptide cleavage produces a 300 nm-long col-lagen triple helix bounded by short terminal pep-tides (telopeptides). These mature collagen molecules are then assembled into fibers, often together with a smaller fraction of molecules of type III, V and XI col-lagens (Figure 7.1).33–35

C H A P T E R

Collagen Structure, Folding and Function

Elena Makareeva and Sergey LeikinEunice Kennedy Shriver National Institute of Child Health and Human Development, National

Institutes of Health, Bethesda, MD, USA

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COLLAGEN TRIPLE HELIX

The distinguishing feature of all collagens is a triple helix formed by three polypeptide chains with a glycine residue in every third position. The obligatory glycines are essential for the triple-helix formation. Gly substi-tution for another amino acid places the side chain of the latter inside the helix core, preventing interchain hydrogen bonding, sterically disrupting the helix and decreasing the helix stability.36,37

X- and Y-positions in Gly-X-Y triplets are occupied by different amino acids that vary along the triple helix. Studies of model peptides revealed that the triple-helix

structure of mammalian collagens is stabilized pri-marily by 4-hydroxyproline (4-Hyp) and Arg in the Y-position as well as by Pro in the X-position.36,38,39 This stabilization is essential for procollagen folding; e.g., deficient hydroxylation of Y-position Pro into 4-Hyp by collagen proline 4-hydroxylases in the absence of ascorbic acid prevents normal synthesis and folding of procollagen.40,41 Other X and Y amino acids affect the triple-helix stability as well, but to a lesser degree.42 Regions with few or no stabilizing X and Y residues are generally less stable, more flexible, may exhibit local unfolding and refolding, and are more susceptible to cleavage by general proteases.43–45 Such flexible regions

FIGURE 7.1 Simplified scheme of type I collagen biosynthesis. (A) C-propeptide folding is assisted by general ER chaperones, including BiP, GRP94, PDI, calreticulin and calnexin; calnexin likely anchors procollagen molecules to the ER membrane. Subsequent folding requires stabili-zation of the forming triple helix by specialized chaperones (HSP47 and potentially other molecules). (B) Gly substitutions and other mutations that reduce the triple-helix stability (and therefore propensity to triple helix formation) result in a folding delay, until triple-helix formation is renucleated on the N-terminal side of the mutation. (C) Folded procollagen is transported from the ER to an ER-Golgi intermediate compart-ment (ERGIC) in large COPII-covered vesicles together with bound HSP47. After dissociation from procollagen in ERGIC/Golgi, HSP47 is trans-ported back to the ER. Procollagen is secreted outside the cell. Its N- and C-propeptides are removed by specialized N- and C-proteinases. This cleavage, which may begin already in the secretory pathway inside the cell, triggers assembly of collagen molecules into fibers.

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might be important for proper formation and function of collagen fibers.44,46,47

Human type I collagen triple helix contains 338 uninterrupted Gly-X-Y repeats within each of the three chains. Pro occupies ~1/3 of X-positions, 4-Hyp occupies ~1/3 of Y-positions, and Arg occupies ~ 1/8 of Y-positions (~80% of Arg residues have Y-position locations). Gly substitutions account for over 80% of severe OI cases.1 Substitutions of X-position Pro, Y-position 4-Hyp, and Y-position Arg also result in OI symptoms,48–54 but sub-stitutions of X-position Arg do not seem to cause OI,52,54–

57 probably because the latter do not destabilize the triple helix. Thus, triple-helix stability and folding appear to play an important role in OI.

Different maps of regions with reduced and increased local triple-helix stability were constructed for type I collagen based on the content of Pro and 4-Hyp residues,58 stability of triple-helical peptides with different sequences,42,59 and stability of the whole col-lagen molecule with Gly substitutions at different sites.60 Approximate locations of the largest regions with higher and lower than average stability based on the latter approach are shown in Figure 7.2A. As discussed in the next section of this review, local triple-helix stability corre-lates with regional variations in the severity of OI caused by Gly substitutions, but this correlation is neither simple nor straightforward.60

Overall, type I collagen triple-helix stability is sur-prisingly low, e.g., the native triple-helical structure of human type I collagen is more thermodynamically favorable than unfolded chains (and therefore stable) only below 34–35°C.63,64 In a physiological environ-ment at normal body temperature, the triple helix is unstable and slowly denatures.63 It is important to emphasize that the apparent triple-helix denaturation temperature (Tm) depends on the heating/equilibration rate and is usually 5–7°C higher than the equilibrium stability of the molecule, because typical measurements allow only for a relatively short equilibration at each temperature.63,65–67

Type I collagen instability appears to have been deliberately designed by Nature, since the apparent Tm value correlates with the normal body temperature for the species from which the protein has been extracted, from arctic fish to mammals.47,68 This instability might be essential for formation of elastic collagen fibers with unique biochemical and mechanical properties.47 Triple-helix denaturation at body temperature is sufficiently slow to allow for N- and C-propeptide cleavage and incorporation of native molecules into fibers,63,64 where the triple helices are stabilized and protected from dena-turation by intermolecular interactions.69 Yet, the low triple-helix stability presents a significant challenge for cells, resulting in a unique folding process that requires highly specialized chaperone machinery.

PROCOLLAGEN FOLDING

Unlike most proteins, which begin to fold from their N-terminal end while the rest of the chain is still being synthesized, procollagen folds from the C-terminal end only after the synthesis of the chains is complete. The folding process begins from association of two pro-α1(I) and one pro-α2(I) chain within the C-propetide region and C-propeptide folding. The next step is folding of the central triple helix, which proceeds from the C- to N-terminal end in a zipper-like manner. N-propeptide folding completes the process. Hydroxylation of most Y-position Pro to 4-Hyp, few specific X-position Pro to 3-hydroxyproline (3-Hyp), and some Lys to hydroxyly-sine (Hyl) as well as glycosylation of some Hyl proceed simultaneously with the synthesis and folding of procol-lagen chains and occur only within unfolded regions of the chains (Figure 7.1A).35–37,70,71

The most unusual step in this process is the triple-helix folding, as it does not conform to the generally accepted protein folding paradigm. In a test tube, when not dis-turbed or helped by other macromolecules, folding or unfolding of a polypeptide chain always proceeds toward more thermodynamically favorable conformations (which have lower free energy at equilibrium).72–74 In the ER, folding of most proteins also follows this general direction. Some non-native interactions between residues (mostly hydrophobic) may be more thermodynamically favorable than native ones (have even lower free energy), resulting in misfolding and/or aggregation of polypeptide chains. To prevent such counterproductive interactions, cells utilize chaperone molecules.75–77 ER chaperones like BiP and GRP94 bind to unfolded and partially folded chains and block counterproductive interactions, e.g., by cycling on and off large hydrophobic patches. Release of these chaperones upon completion of the folding pro-cess serves as one of the signals that the protein can be transported out of the ER toward its final destination.75 Misfolding causes sequestration of BiP and GRP94 on misfolded chains. The resulting release of these chaper-ones from their receptors on the ER membrane activates conventional unfolded protein response (UPR) signal-ing.78,79 For most proteins, preferential chaperone binding makes unfolded and partially folded chain conformations less thermodynamically unfavorable, but not more favor-able than the native state.

For procollagen, however, the folding scenario is drastically different. Cells have to utilize specialized chaperones that make the folding thermodynamically favorable by preferentially binding to the native triple helix rather than unfolded chains.80 Without such chap-erones, human procollagen folding is favorable below 34–35°C, but unfavorable above this temperature.64 At 37–38°C, native procollagen denatures within sev-eral days and remains unfolded, because its unfolded

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conformation is more favorable than the folded one. BiP and GRP94 appear to be required for procolla-gen C-propeptide folding,70 but their binding to the triple-helical region of procollagen chains would be counterproductive, making folding of this region even more unfavorable.80 One of the chaperones that

make the triple-helix folding favorable appears to be HSP47.61,64,81,82 Other potential candidates include FKBP6583 and a complex of CRTAP, prolyl 3-hydrohylase 1 (P3H1) and a peptidyl-prolyl isomerase cyclophilin B (CYPB),84 but their role in procollagen folding is less clear.80 For instance, in vitro studies suggested that

FIGURE 7.2 Collagen triple-helix folding and OI severity. (A) HSP47 binding sites and large regions with low (flex regions) and high (anchor regions) triple-helix stability/propensity in human type I collagen. HSP47 binding sites were mapped from the sequences reported for triple-helical peptides.61 The map of low and high stability regions updates the previously published one60 based on ΔTm measurements for additional Gly substitutions. Less stable regions with fewer HSP47 binding sites are the most likely bottlenecks for the procollagen folding process. (B) Severity scores for Gly-to-Ser substitutions in the α1(I) chain based on patient phenotypes reported in the online OI variant database54 prior to May 1, 2012. Individual substitution scores (filled circles) were assigned as follows: mild mutations reported as type I OI were given the score 1, type I/IV OI – score 1.5, type IV OI – score 2, type III/IV OI – score 2.5, type III OI – score 3, type II/III OI – score 3.5, and most severe type II OI – score 4. Averaged scores for substitution sites (open circles) were calculated by averaging the scores for all substitutions at the same site and then averaging the resulting values for sites located within five Gly-X-Y triplets. The five triplet size of the “running average” window was based on the correlation length of fluctuations in the triple-helix structure estimated from H–D exchange experiments.60 Similar scores were obtained for running average window sizes from three to eight triplets. (C) Averaged severity scores for Gly substitutions in the α1(I) chain. Open circles show Gly-to-Ser substitutions (same as in panel B), the bold line shows all substitutions pooled together without distinguishing the identity of the substituting residue. Comparison of the two plots reveals three regions of extremely severe/lethal mutations: (1) from ~250 to ~300; (2) from ~700 to ~800; and (3) from ~900 to ~950. On the N-terminal side, these regions border/overlap with N-flex, M-flex2, and C-flex regions. Reduced triple-helix stability and low density of HSP47 binding sites within the latter regions should hinder re-nucleation of triple-helix folding past the mutation sites (cf., Figure 7.1), potentially explaining the increased mutation severity. (D) Averaged severity scores for Gly substitutions in the α2(I) chain. The peaks with increased severity correspond to previously described clusters of lethal mutations.62 However, these peaks might result from clustering of more severe Gly-to-Asp, Glu, Arg, and Val substitutions and not from effects of the substitution location, since less severe Gly-to-Ser substitutions do not show the same pattern as all Gly substitutions pooled together. Overall, regional severity variations for Gly substitutions in the α2(I) chain appear to be different from the α1(I) chain, but the reported set of mutations does not appear to be large enough to establish a reliable pattern.

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FKBP65 might act as such a chaperone,83 but FKBP65 deficiency does not seem to delay procollagen folding in vivo.85 For additional discussion of potential roles of different ER proteins in procollagen folding, we refer the reader to the preceding chapter in this book.

The response of cells to procollagen misfolding is also distinct. C-propeptide misfolding causes sequestration and upregulation of BiP, activates UPR signaling, and leads to ER associated degradation (ERAD) of misfolded chains by proteasomes.86–88 Yet, triple-helix misfolding does not trigger these conventional responses.86,89–91 Instead, misfolded procollagen forms insoluble aggre-gates, which are delivered to lysosomes by autophagy and degraded there.91 Similar features were observed in so-called ER overload response to misfolding and aggregation of some proteins from the serine protease inhibitor (serpin) family,92 but the ER stress response to procollagen triple-helix misfolding is still poorly understood and might not involve the same signaling pathways.91,93

Substitutions of obligatory Gly, X-position Pro, Y-position 4-Hyp, and Y-position Arg residues make procollagen folding more difficult, since they further destabilize the triple helix. For instance, Gly substitu-tions reduce the apparent triple-helix Tm by ΔTm~2°C on average; and some reduce it as much as ~ 5°C.60 Cells manage to fold these molecules anyway, prob-ably by upregulating the corresponding chaperones. For instance, HSP47 upregulation was reported for Gly substitutions.94 Still, triple-helix destabilization increases procollagen misfolding, results in retention of misfolded molecules in the ER, increases ER stress, and thereby affects the function of collagen-producing cells.

ER stress response to procollagen misfolding might have particularly severe consequences for osteoblasts, since these cells produce massive amounts of type I collagen. Hence, ER stress has been proposed to be a major factor determining the severity of OI.80,89,90,95,96 However, not only the mechanism of this response, but also the extent of the underlying ER stress is still poorly characterized, even for the most common and best stud-ied OI cases caused by Gly substitutions. The complex-ity of the relationship between such substitutions and the resulting procollagen misfolding may be illustrated on the example of ΔTm, which is a measure of the colla-gen triple-helix destabilization by the mutation.

One is intuitively tempted to use ΔTm as a proxy for the extent of procollagen misfolding, but such an assumption turns out to be wrong. There is actually no simple relationship or correlation between the ΔTm value and the extent to which procollagen folding is disrupted by the substitution. For instance, Gly sub-stitutions with Arg, Glu, Asp or Val cause much larger disruptions in the triple-helix folding and structure near the mutation site than substitutions with Ala or Ser.97,98

As a result, Arg, Glu, Asp and Val substitutions should cause longer folding delays, more extensive misfold-ing and accumulation of procollagen in the ER, as well as more severe ER stress than Ala and Ser substitutions. However, all of these mutations produce similar ΔTm when they occur at the same location within the triple helix.60,99 This seeming discrepancy may be explained as follows. The value of ΔTm depends on the contribution of a region surrounding the mutation site to the stabil-ity of the whole triple helix.60 In contrast, the extent of procollagen folding disruption depends on the abil-ity of the triple-helix folding process to accommodate the substitution and proceed through the mutation site.98,100 When a Gly substitution is encountered, the zipper-like folding process pauses until the triple helix is re-nucleated on the N-terminal side of the mutation.37,101 The re-nucleation efficiency is affected by the location of Gly-X-Y triplets with high triple-helix propensity and stability and HSP47 binding sites on the N-terminal side of the mutation as well as by the ability of the helix to accommodate the mutation. In particular, a Gly substitu-tion on the C-terminal side of a large region with low tri-ple-helix stability that has few or no HSP47 binding sites is likely to cause a major disruption of procollagen fold-ing and accumulation of misfolded molecules in the ER. At the same time, such a mutation might cause smaller than average ΔTm because of the minimal contribution of this region to the stability of the whole triple helix. For additional discussion of Gly substitution effects on the triple-helix structure and folding, see other chapters in this book.

Despite these complexities, the expected severity of ER stress caused by procollagen misfolding might explain at least some of the observed trends in the sever-ity of OI caused by Gly substitutions, as illustrated in Figure 7.2. Indeed, panels C and D in Figure 7.2 show that Gly substitutions tend to be more severe in the α1(I) chain compared to the α2(I) chain, which might be explained by a higher fraction of molecules containing mutant α1(I) chains (heterozygous mutations in the α1(I) and α2(I) chains are expected to produce 75 and 50% molecules with mutant chains, respectively, since chain association at the C-propeptide should not be affected by triple-helix mutations). More severe OI caused by Gly substitutions with Arg, Glu, Asp or Val compared with Ala or Ser might be explained by larger disrup-tions in the triple-helix folding and structure introduced by the former.97,102 The lack of apparent correlations of OI severity with ΔTm

62 might be explained by the lack of a simple relationship between ΔTm and the disrup-tion of procollagen folding.60 Milder OI phenotypes of substitutions near the N-terminal end of the triple helix might be explained by weaker ER stress response to delayed folding of a shorter part of the triple helix, since such molecules are likely to have lower probability of

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aggregation. In addition, very high triple-helix stabil-ity and propensity within the N-anchor region103 might enhance triple helix re-nucleation on the N-terminal side of closely located mutations. Three pronounced peaks in the severity of α1(I) Gly substitutions (observed both for all Gly substitutions pooled together and just for Gly-to-Ser) might be explained by the lack of triple-helix re-nucleation sites and a reduced number of HSP47 bind-ing sites on the N-terminal side of these peaks.

PROCOLLAGEN TRAFFICKING

Export of procollagen from the ER and its trafficking through the cell also appear to involve distinct mecha-nisms. In particular, secretion of incompletely folded procollagen by cells that produce no or dysfunctional HSP47 could indicate HSP47 involvement in control-ling the quality of procollagen folding in the ER.10,104 However, HSP47 cannot prevent the export of incom-pletely folded chains from the ER by preferential bind-ing to and tagging them like general ER chaperones bind to and tag incompletely folded chains of other pro-teins. Instead, HSP47 preferentially binds to and is co-transported with natively folded procollagen from the ER to Golgi.81,82,105 Thereby, it is more likely to promote the transport of native procollagen rather than inhibit the transport of misfolded chains. Secretion of incom-pletely folded procollagen by HSP47 knockout cells might result simply from inability of these cells to fold any procollagen molecules, some of which make their way out of the cells. Furthermore, unfolded pro-α2(I) chains seem to travel from the ER to Golgi in cells that have no chaperone deficiencies. In particular, knockout of pro-α1(I) chains prevents the association and fold-ing of pro-α2(I) chains, resulting in translocation of unfolded pro-α2(I) chains to Golgi where they are tar-geted for lysosomal degradation via an unknown path-way.106 Transfection of cells with GFP-tagged pro-α2(I) chains (most of which are not expected to incorporate into folded procollagen trimers107) results in accumula-tion of such chains in Golgi cisternae.108

Other chaperones involved in the quality control of procollagen chain folding in the ER include BiP and PDI. BiP binding likely prevents the export of chains with misfolded C-propeptides.86–88 PDI binding seems to prevent the export of molecules with large structural defects, such as truncated chains.109 Yet, the overall pic-ture of procollagen folding quality control at export from the ER is still largely unclear.96

Like other proteins, procollagen is transported from the ER to Golgi in COPII-covered vesicles,110 but procol-lagen might require distinct adaptor protein(s) for load-ing into these vesicles. Studies of MIA3 knockout cells and animals suggest that this ER membrane protein

might play such a role, but it is not clear yet whether MIA3 is essential for trafficking of all collagens or pri-marily type VII collagen.111–113

In addition to distinct adaptor(s), trafficking of 300 nm-long procollagen molecules requires larger than usual (60–80 nm) vesicles. Mono-ubiquitylation of COPII pro-tein SEC31 by CUL3-KLHL12 ubiquitin ligase complex has been recently reported to be essential for formation of such vesicles.114 Suppression of this ubiquitylation impairs export of type I procollagen from the ER, although this process might be a general rather than a collagen-specific regulator of the COPII-covered vesicle size.

Another interesting aspect of procollagen traffick-ing is that N- and C-propeptide cleavage might begin already in the secretory pathway inside the cell.115 The N-propeptide might be cleaved already in cis-Golgi while C-propeptide cleavage appears to begin only in post-Golgi compartments.116 Nevertheless, a significant fraction of procollagen molecules is secreted with intact N- and C-propeptides, at least in cell culture.34,115–117

These advances clarify some of the unique features of procollagen trafficking, but they do not explain how cells distinguish procollagen molecules with Gly sub-stitutions and selectively retain them in the ER for sub-sequent degradation. Such retention, which has been reported in a number of studies,89,118,119 might be a cru-cial factor in excessive ER stress and osteoblast mal-function in OI.80 We hope that future studies of mutant procollagen trafficking will address this problem.

ASSEMBLY AND FUNCTION OF COLLAGEN FIBERS

Correctly folded collagen molecules are capable of unassisted self-assembly into fibers once their N- and C-propeptides are cleaved.120 Yet, fiber assem-bly (fibrillogenesis) is tightly regulated in vivo by a vari-ety of molecules, e.g., it occurs on the outer cell surface although N- and C-propeptide cleavage begins inside the cell.116,121 In bone, collagen fibers provide a template for deposition of hydroxyapatite mineral, glue min-eral crystals together, mechanically reinforce the tissue (akin to steel bars in concrete), and bind other molecules essential for normal bone matrix homeostasis and min-eralization. Dysregulation of assembly, deposition, and function of collagen fibers in bone might be caused by (1) insufficient collagen synthesis; (2) abnormal osteo-blast function; (3) abnormal collagen–collagen interac-tions; and (4) abnormal collagen interactions with other molecules or minerals. However, relative contributions of these abnormalities are often difficult to distinguish.

For instance, insufficient collagen synthesis is consid-ered to be responsible for the mild (type I) OI in patients with null-allele COL1A1 mutations (which prevent

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translation of this allele, e.g., due to RNA degradation).1 Yet, these mutations should also disrupt the balance between the pro-α1(I) and pro-α2(I) chains in the ER. Excess pro-α2(I) chains are transported through Golgi to lysosomes for degradation,106 but intracellular accumu-lation of these chains and the increased need for their degradation might affect osteoblast differentiation and function.

Disruption of osteoblast differentiation and function by procollagen misfolding and retention in the ER was documented in the Aga2 mouse with a C-propeptide mutation95 and Brtl mouse with a Gly substitution in the α1(I) chain.122,123 In the Aga2 mouse, one might expect osteoblast malfunction to be the primary cause of pathology since most of the mutant molecules appear to be retained and degraded by cells.95 Yet, even inefficient secretion of these mutant molecules could affect fiber formation and function by altering collagen interactions, because such molecules are likely to be overmodified and their defective C-propeptides might not be prop-erly cleaved. In the Brtl mouse, the triple-helix muta-tion might be expected to affect collagen interactions, but measurements of collagen–collagen interactions in tendons and in vitro fibrillogenesis experiments reveal no such effects.124 Instead, studies of collagen-producing cells point to osteoblast malfunction as a potential main contributing factor.89,122,123 Normalization of the bone structure and properties by 1–2% engraftment of trans-planted wild-type cells further support this hypothe-sis.125 The donor cells produce ~10 times more collagen than their fraction of total osteoblast population, empha-sizing rather dramatic malfunction of host osteoblasts. Based on these and other observations, disruption of osteoblast differentiation and function seems to be an important factor in most OI cases caused by mutations that affect procollagen folding.80

Disruptions in collagen fiber formation, structure and function by uncleaved propeptides were reported for several mutations in the triple helix as well as for muta-tions within propeptide cleavage sites and enzymes, but not all of these disruptions were found to contribute sig-nificantly to bone pathology. In particular, incorporation of molecules with uncleaved N-propeptides into collagen fibers alters the fiber size, structure and strength.126–129 Mutations in the N-propeptide cleavage site and in the N-proteinase that cleaves at this site lead to Ehlers–Danlos syndrome (EDS) type VII with severe joint hyperextensibility and skin fragility, but only mild or no manifestations of bone pathology.127,130–132 Mutations in the N-anchor region of the triple helix cause EDS VII-like joint hyperextensibility in combination with moder-ately severe OI.133 Apparently, disruption of procollagen folding and osteoblast function by N-anchor mutations produces OI symptoms and disruption of N-propeptide cleavage produces EDS symptoms.103,133 Mutations in

the C-propeptide cleavage site and the corresponding C-proteinase (BMP1) alter collagen–mineral interactions and cause OI with higher than expected bone mineral density.15,134,135 Collagen fiber mineralization in these patients is likely disrupted by incorporation of molecules with uncleaved C-propeptides.135

Abnormal collagen–collagen interactions were docu-mented for PLOD213,136 and FKBP1085 mutations, which produced overlapping OI phenotypes11–14,137 and similar deficiency in collagen telopeptide lysyl hydroxylation85 by lysyl hydroxylase 2 (LH2) encoded by PLOD2. The telopeptide lysyl hydroxylation deficiency prevents covalent crosslinking between adjacent molecules in col-lagen fibers.138 Dysregulated collagen fiber deposition in cultures of dermal fibroblasts from OI patients with FKBP10 mutations also appears to be a consequence of the deficient crosslinking.85 Yet, FKBP65 encoded by FKBP10 is an ER chaperone83,139 and stress-response pro-tein involved in Ca2+ signaling,140 so that FKBP10 muta-tions might cause abnormal collagen biosynthesis and/or malfunction of osteoblasts as well. PLOD2 mutations might do the same by altering FKBP65 functions, since abnormal lysyl hydroxylation in FKBP65-deficient cells points to an interaction between FKBP65 and LH2.85

Aberrant regulation of collagen fibrillogenesis by extracellular matrix molecules might underlie OI caused by SERPINF1 mutations. Pigment epithelium derived factor (PEDF) encoded by SERPINF1 is a secreted col-lagen-binding protein141 that does not seem to affect collagen biosynthesis16 but is a potent fibrillogenesis inhibitor.142 It is capable of significantly slowing down fiber formation at physiological concentrations by bind-ing to the triple helix and blocking collagen–collagen interactions (Konopko et  al., unpublished results), so that it might be an important negative fibrillogenesis regulator in vivo. PEDF might affect bone formation and quality, e.g., by preventing excessive deposition of dis-organized collagen fibers, which would explain why PEDF deficiency causes OI in humans16,17,143 but not in mice.144,145 (Collagen fibers are less well organized and their formation might be less tightly regulated in mouse bones; and fibrillogenesis of mouse collagen occurs under much more permissive conditions compared to human collagen.) The delayed onset of bone pathology in type VI OI patients146 caused by PEDF deficiency17 might also be explained by runaway fibrillogenesis, which is likely to be more detrimental for the deposition of better organized lamellar bone during remodeling than for the deposition of woven bone in early develop-ment. Yet, PEDF has been reported to affect the function of osteoblasts and osteoclasts too.147,148

One of the most complex scenarios for the analysis of different contributions to bone matrix dysregulation in OI is exemplified by CRTAP, LEPRE1 and PPIB muta-tions. Prolyl-3-hydroxylase 1 (P3H1) encoded by LEPRE1

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is responsible for converting proline in specific X-positions of the collagen triple helix into 3-hydroxyproline (3-Hyp).149,150 CRTAP protein forms a complex with P3H1149 and is essential for P3H1 stability and function in the ER.2,151 CYPB encoded by PPIB is a peptidyl prolyl isomerase, which is believed to be essential for accelerat-ing procollagen triple-helix folding152,153 and might be involved in procollagen trafficking in the cell.154 In addi-tion, CYPB forms a complex with CRTAP and P3H1,84,149 complexes with other ER chaperones155–158 and appears to be involved in a variety of processes within ER.5,75 Functional null mutations in CRTAP, LEPRE1 and PPIB were found to delay procollagen folding3,4,158,159 and result in excessive accumulation of procollagen in the ER.5 Dysregulated deposition of collagen fibers caused by these mutations5,150,158,160,161 might thus be associated with abnormal differentiation and function of collagen-produc-ing cells. However, the lack of essential 3-Hyp residues in the triple helix might affect collagen–collagen interac-tions and thereby fibrillogenesis as well.162 It might also affect collagen interactions with other molecules that are essential for normal fiber formation and function in bone. Finally, CYPB deficiency or abnormal CYPB homeosta-sis in the ER due to CRTAP and P3H1 deficiencies might contribute to dysregulation of collagen fiber formation by affecting procollagen trafficking or by affecting cell func-tions due to CYPB roles in the ER unrelated to collagen.

DYSREGULATION OF TYPE I COLLAGEN HOMEOSTASIS BY GLY SUBSTITUTIONS

To integrate different concepts of type I collagen homeostasis discussed above and further illustrate their implications, it is useful to discuss models for genotype–phenotype relationships in OI caused by Gly substitu-tions in the triple helix. The simplest model correlates OI severity with the identity of the amino acid that substi-tutes for Gly. Amino acids that cause more severe disrup-tions in the structure and stability of the triple helix are expected to be more detrimental for procollagen fold-ing, osteoblast function, and assembly and function of collagen fibers, thereby resulting in more severe OI. This concept is discussed in more detail in another chapter. Here, we briefly review other models, which address correlations of OI severity with the substitution location within different regions of the triple helix.

The gradient model takes into account the C-to-N terminal direction of the triple-helix folding and pre-dicts increased severity of more C-terminal muta-tions, for which more extensive overmodification is expected.163–165 However, such a severity gradient is observed only within the first 200 N-terminal amino acids (Figure 7.2) and the severity of OI does not appear to correlate with the overmodification.62,166

Another model assumes that OI severity might be affected by variations in the local stability of the triple helix at the substitutions sites.167 The apparent lack of correlations between regional variations in severity of Gly substitutions and different maps of local triple-helix stability might be viewed as an argument against this idea.62 Yet, this lack of correlations might simply indicate that local triple-helix stability at the substitution site is only one of several factors determining the effect of the substitution on procollagen folding (see “Procollagen folding,” above and ER stress model, below).

Binding site disruption models associate regional variations in Gly substitution severity with dysregula-tion of collagen interactions with collagen, other matrix molecules, and cell surface receptors caused by disrup-tion of the corresponding binding sites.62,168–170 In our opinion, these models have two major weaknesses: first, one might then expect a large number of OI patients with substitutions that disrupt the same binding sites but do not involve Gly, X-position Pro, Y-position 4-Hyp, or Y-position Arg, which is not the case. Second, these models do not explain why the severity patterns for Gly substitutions in the α1(I) and α2(I) chains are completely different (Figure 7.2). Since triple-helix disruptions caused by similar Gly substitutions in the α1(I) and α2(I) chains are similar in most cases,60 one might expect α1(I) and α2(I) mutations to have similar effects on collagen interactions. The idea that collagen interactions depend more on the specific α2(I) chain sequence than on the triple-helix structure seems to be inconsistent with the lack of OI patients with amino acid substitutions that do not disrupt the triple-helix structure.

In fact, we can think of only one explanation for dif-ferent regional severity patterns of α1(I) and α2(I) muta-tions, which is a difference in the triple-helix disruption in molecules with two vs. one mutant chain. Since mol-ecules with two mutant α1(I) chains account for only 25% of type I collagen produced by cells, they are unlikely to change collagen interactions in the extracellular matrix qualitatively. At the same time, their abnormal folding, selective retention and accumulation in the ER might qualitatively alter osteoblast function.

The last idea brings us to the ER stress model, which attributes variations in OI severity to differ-ences in osteoblast malfunction in response to ER stress caused by procollagen misfolding. As we discussed in “Procollagen folding,” above, this model is con-sistent with the observed regional severity variations (Figure 7.2), reduced severity of N-terminal mutations, increased severity of Gly substitutions in the α1(I) chain and effects of the identity of the substituting amino acid. Furthermore, it might explain phenotypic variations observed for the same mutation in different patients62 by variability of patient osteoblast adaptation to ER stress. Although discussed for many years, the ER stress

79

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model has not been considered a prime contender for explaining bone pathology caused by Gly substitutions, because no upregulation of BiP by these mutations has been detected.86 BiP upregulation is a commonly used indicator of canonical UPR signaling activation in ER stress response to protein misfolding,78 but the ER stress response to misfolding of procollagen triple helix might be different.80 Recent studies show that aggregation of some misfolded proteins,171 including procollagen with Gly substitutions,91 might trigger a different pathway of ER stress response, which does not involve upregulation of BiP and activation of UPR. Severe dilation of fibroblast ER cisternae in Brtl mouse skin89 without BiP upregula-tion90 confirms that this ER stress response is likely to be an important factor in at least some OI cases.

CONCLUSION

In summary, the relationship between type I collagen biology and OI pathophysiology might be represented by categorizing the latter based on disruptions in type I collagen homeostasis as: (1) insufficient collagen pro-duction; (2) extracellular collagen malfunction; and (3) osteoblast malfunction resulting from abnormal pro-collagen folding and/or trafficking. We must stress that these disruptions are interrelated and all of them are likely to contribute to OI. By somewhat reductionist discussion of the corresponding concepts, we intend only to highlight the aspects of type I collagen biology that might be important for better understanding OI and developing therapeutic strategies for this devastat-ing disorder. Our emphasis on procollagen folding and ER stress stems from the belief that targeting osteoblast malfunction offers the best hope for new pharmaco-logical treatments of the most common forms of severe OI, providing an alternative to much more challenging gene therapy and stem cell transplantation. We hope that the present review makes the case for a more care-ful analysis of this idea.

AcknowledgmentThis work was funded by the Intramural Research Program, NICHD, NIH.

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