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Mapping the Ligand-binding Sites and Disease-associated Mutations on the Most Abundant Protein in the Human, Type I Collagen*

Open AccessPublished:November 09, 2001DOI:https://doi.org/10.1074/jbc.M110709200
      Type I collagen is the most abundant protein in humans, and it helps to maintain the integrity of many tissues via its interactions with cell surfaces, other extracellular matrix molecules, and growth and differentiation factors. Nearly 50 molecules have been found to interact with type I collagen, and for about half of them, binding sites on this collagen have been elucidated. In addition, over 300 mutations in type I collagen associated with human connective tissue disorders have been described. However, the spatial relationships between the known ligand-binding sites and mutation positions have not been examined. To this end, here we have created a map of type I collagen that includes all of its ligand-binding sites and mutations. The map reveals the existence of several hot spots for ligand interactions on type I collagen and that most of the binding sites locate to its C-terminal half. Moreover, on the collagen fibril some potentially relevant relationships between binding sites were observed including the following: fibronectin- and certain integrin-binding regions are near neighbors, which may mechanistically relate to fibronectin-dependent cell-collagen attachment; proteoglycan binding may potentially impact upon collagen fibrillogenesis, cell-collagen attachment, and collagen glycation seen in diabetes and aging; and mutations associated with osteogenesis imperfecta and other disorders show apparently nonrandom distribution patterns within both the monomer and fibril, implying that mutation positions correlate with disease phenotype. These and other observations presented here may provide novel insights into evaluating type I collagen functions and the relationships between its binding partners and mutations.
      OI
      osteogenesis imperfecta
      PG
      proteoglycan
      KSPG
      keratan sulfate PG
      GAG
      glycosaminoglycan
      DDR
      discoidin domain receptor
      COMP
      cartilage oligomeric matrix protein
      OPO
      osteoporosis
      OPA
      osteopenia
      DSPG
      dermatan sulfate PG
      CSPG
      chondroitin sulfate PG
      HSPG
      heparan sulfate PG
      Type I collagen is ubiquitous in all vertebrates and is among the largest and most complex of all macromolecules. It is synthesized as a soluble, procollagen form, composed of globular C- and N-propeptides, joined to their respective ends of the triple helix (
      • Prockop D.J.
      • Kivirikko K.I.
      ). Type I procollagen (Fig. 1A) is composed of two α1 and one α2 chains, each of about 1000 amino acids. Upon secretion from the cell the propeptides are cleaved by the C- and N-proteinases, and the collagen monomer is assembled into the collagen fibril (Fig. 1,A and B), which is the form of the protein found in tissues (Fig. 1C), and consists of microfibrils, or 5-mer bundles of overlapping monomers. Adjacent monomers overlap each other by 234 residues, giving rise to the 67-nm wide “D-period,” the basic repeat structure of the fibril, seen in electron micrographs of collagen fibrils (Fig. 1C). Each D-period consists of an “overlap” and a “gap” zone, which appear as alternating light and dark bands in negatively stained preparations or as a pattern of fine cross-fibril bands (called “a,” “b,” “c,” “d,” and “e” bands) in positively stained preparations (
      • Chapman J.A.
      ).
      Figure thumbnail gr1
      Figure 1Type I collagen monomeric and fibrillar structure. A, by rotary shadowing electron microscopy, procollagen (≅300 nm long) appears as a rope-like triple helix to which globular C-terminal (to the right) and N-terminal domains are attached. B, Chapman's model (
      • Chapman J.A.
      ) of the collagen fibril. The boxed region includes one D-period repeat comprised of the type I collagen telopeptides and complete triple helical sequences, which are shown in detail in Fig. .C, appearance of the type I collagen fibril visualized by negative and positive staining and transmission electron microscopy (see text for description). The arrow indicates the left border of the overlap zone. This fibril preparation was used to localize heparin-binding sites; thus, heparin-gold particles appear asdark circles bound to the fibril. a–e, cross-fibril bands. Reproduced from The Journal of Cell Biology, 1994, 125, 1179–1188 by copyright permission of The Rockefeller University Press (
      • San Antonio J.D.
      • Lander A.D.
      • Karnovsky M.J.
      • Slayter H.S.
      ).
      Type I collagen is proposed to be a key structural component of load-bearing tissues such as bones, tendons, and ligaments; however, it likely also plays many other physiologic functions. Thus, over the past 30 years various molecules and cells have been described that exhibit specific interactions with type I collagen, and for about half of these, their sites of interaction with the collagen triple helix and fibril have been mapped (Tables I andII). These include, for example, sequences proposed to mediate cell adhesion, binding to other matrix molecules, interactions with tissue calcification factors, and glycation, a process of glucose adduct formation onto protein, proposed to contribute to pathologies associated with diabetes and aging (
      • Reiser K.M.
      • Amigable M.A.
      • Last J.A.
      ,
      • Hadley J.C.
      • Meek K.M.
      • Malik N.S.
      ). In addition, over 300 mutations in type I collagen leading to OI1 and other connective tissue disorders have been described (partial lists are found in Refs.
      • Dalgleish R.
      ,
      • Ward L.M.
      • Lalic L.
      • Roughley P.J.
      • Glorieux F.H.
      ,
      • Pallos D.
      • Hart P.S.
      • Cortelli J.R.
      • Vian S.
      • Wright J.T.
      • Korkko J.
      • Brunoni D.
      • Hart T.C.
      ); additional unpublished mutations are included here).
      Table ICollagen interactive molecules/functional domains, mapped
      Molecule/interactionReference
      α1β1 integrin
      • Knight C.G.
      • Morton L.F.
      • Onley D.J.
      • Peachey A.R.
      • Messent A.J.
      • Smethurst P.A.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      ,
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ,
      • Knight C.G.
      • Morton L.F.
      • Peachey A.R.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      α2β1 integrin
      • Knight C.G.
      • Morton L.F.
      • Onley D.J.
      • Peachey A.R.
      • Messent A.J.
      • Smethurst P.A.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      ,
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ,
      • Staatz W.D.
      • Walsh J.J.
      • Pexton T.
      • Santoro S.A.
      ,
      • Knight C.G.
      • Morton L.F.
      • Peachey A.R.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      Amyloid protein precursor
      • Beher D.
      • Hesse L.
      • Masters C.L.
      • Multhaup G.
      Angiogenesis inhibition
      • Sweeney S.M.
      • Guy C.A.
      • Fields G.B.
      • San Antonio J.D.
      COMP
      • Rosenberg K.
      • Olsson H.
      • Morgelin M.
      • Heinegard D.
      Chondroitin sulfates
      • Scott J.E.
      • Glanville R.W.
      Collagen V
      • Niyibizi C.
      • Eyre D.R.
      C-proteinase
      • Ayad S.
      • Boot-Handford R.P.
      • Humpries M.J.
      • Kadler K.E.
      • Shuttleworth C.A.
      The Extracellular Matrix Facts Book.
      C-telopeptide fibrillogenesis nucleation
      • Prockop D.J.
      • Fertala A.
      Decorin
      • Yu L.
      • Cummings C.
      • Sheehan J.K.
      • Kadler K.E.
      • Holmes D.F.
      • Chapman J.A.
      ,
      • Fleischmajer R.
      • Fisher L.W.
      • MacDonald E.D.
      • Jacobs Jr., L.
      • Perlish J.S.
      • Termine J.D.
      Decorin core
      • Keene D.R.
      • San Antonio J.D.
      • Mayne R.
      • McQuillan D.J.
      • Sarris G.
      • Santoro S.A.
      • Iozzo R.V.
      Dermatan sulfates
      • Scott J.E.
      • Glanville R.W.
      Fibrillogenesis inhibition
      • Prockop D.J.
      • Fertala A.
      Fibroblast adhesion
      • Grab B.
      • Miles A.J.
      • Furcht L.T.
      • Fields G.B.
      Fibronectin
      • Kleinman H.K.
      • McGoodwin E.B.
      • Dzamba B.J.
      • Wu H.
      • Jaenisch R.
      • Peters D.M.
      Glycation, α1(I)CNBr3 peptide
      • Reiser K.M.
      • Amigable M.A.
      • Last J.A.
      Glycation, α2(I)CNBr3–5 peptides
      • Reiser K.M.
      • Amigable M.A.
      • Last J.A.
      Glycation, fibril
      • Hadley J.C.
      • Meek K.M.
      • Malik N.S.
      Heparin
      • San Antonio J.D.
      • Lander A.D.
      • Karnovsky M.J.
      • Slayter H.S.
      ,
      • Sweeney S.M.
      • Guy C.A.
      • Fields G.B.
      • San Antonio J.D.
      Integrins, denatured collagen
      • Dedhar S.
      • Ruoslahti E.
      • Pierschbacher M.D.
      ,
      • Gullberg D.
      • Gehlsen K.R.
      • Turner D.C.
      • Ahlen K.
      • Zijenah L.S.
      • Barnes M.J.
      • Rubin K.
      Interleukin-2
      • Somasundaram R.
      • Ruehl M.
      • Tiling N.
      • Ackermann R.
      • Schmid M.
      • Riecken E.O.
      • Schuppan D.
      Intermolecular cross-link
      • Silver F.H.
      • Trelstad R.L.
      ,
      • Zimmermann B.K.
      • Pikkarainen J.
      • Fietzek P.P.
      • Kuhn K.
      Keratan sulfates
      • Scott J.E.
      • Glanville R.W.
      MMPs 1, 2, and 13 cleavage
      • Lauer-Fields J.L.
      • Tuzinski K.A.
      • Shimokawa K.
      • Nagase H.
      • Fields G.B.
      N-proteinase
      • Ayad S.
      • Boot-Handford R.P.
      • Humpries M.J.
      • Kadler K.E.
      • Shuttleworth C.A.
      The Extracellular Matrix Facts Book.
      Phosphophoryn
      • Traub W.
      • Jodaikin A.
      • Arad T.
      • Veis A.
      • Sabsay B.
      ,
      • Dahl T.
      • Sabsay B.
      • Veis A.
      Thrombospondin
      • Galvin N.J.
      • Vance P.M.
      • Dixit V.M.
      • Fink B.
      • Frazier W.A.
      Vertebrate collagenase cleavage
      • Wu H.
      • Byrne M.H.
      • Stacey A.
      • Goldring M.B.
      • Birkhead J.R.
      • Jaenisch R.
      • Krane S.M.
      The sites of interaction of the molecules on type I collagen were mapped in various ways. In some cases, ligands were bound to the collagen I monomer, e.g. phosphophoryn and COMP, and examined by rotary shadowing electron microscopy. In other cases, binding regions on the fibril were visualized using antibodies or stains specific for various collagen interactive ligands and examined by transmission electron microscopy. Gross regions of interaction, such as those of the amyloid protein precursor, one of the α2(I) chain α2β1 integrin binding sites, and the interleukin-2 binding sites, were determined by studying ligand interactions with CNBr peptides of the type I collagen α1(I) or α2(I) chains. All other sites were determined using various type I collagen mimetic peptides. MMP, matrix metalloproteinase.
      Table IICollagen Interactive molecules/cells, not mapped
      Molecule/interactionReference
      Acetaldehyde
      • Wess T.J.
      • Wess L.
      • Miller A.
      αVβ3 integrin
      • Davis G.E.
      Antiplatelet protein (from leech Haementeria officinalis)
      • Depraetere H.
      • Kerekes A.
      • Deckmyn H.
      Biglycan
      • Hunzelmann N.
      • Anders S.
      • Sollberg S.
      • Schonherr E.
      • Krieg T.
      Bilirubin
      • Kapoor C.L.
      BM40/SPARC (secreted protein acidic and rich in cysteine)/osteonectin
      • Sasaki T.
      • Hohenester E.
      • Gohring W.
      • Timpl R.
      ,
      • Termine J.D.
      • Kleinman H.K.
      • Whitson S.W.
      • Conn K.M.
      • McGarvey M.L.
      • Martin G.R.
      Calcium-binding proteins MRP8 and -14
      • Mahnke K.
      • Bhardwaj R.
      • Sorg C.
      Calin (from leech Hirudo medicinalis)
      • Depraetere H.
      • Kerekes A.
      • Deckmyn H.
      Collagen-associated cornea molecules
      • Underwood P.A.
      • Bennett F.A.
      • Mott M.R.
      • Strike P.
      DDR1 and DDR2
      • Shrivastava A.
      • Radziejewski C.
      • Campbell E.
      • Kovac L.
      • McGlynn M.
      • Ryan T.E.
      • Davis S.
      • Goldfarb M.P.
      • Glass D.J.
      • Lemke G.
      • Yancopoulos G.D.
      • Vogel W.
      • Gish G.D.
      • Alves F.
      • Pawson T.
      Entamoeba histolyticacollagen binding proteins
      • Rosales-Encina J.L.
      • Campos-Salazar M.S.
      • Rojkind Matluk M.
      Fibromodulin
      • Hedbom E.
      • Heinegard D.
      Gla protein
      • Lawton D.M.
      • Andrew J.G.
      • Marsh D.R.
      • Hoyland J.A.
      • Freemont A.J.
      Glycoprotein 46
      • Clarke E.P.
      • Cates G.A.
      • Ball E.H.
      • Sanwal B.D.
      Heat shock protein 47
      • Razzaque M.S.
      • Nazneen A.
      • Taguchi T.
      Lumican
      • Rada J.A.
      • Cornuet P.K.
      • Hassell J.R.
      Lymphocyte membrane molecules
      • Arencibia I.
      • Hauzenberger D.
      • Sundqvist K.G.
      Myelin-associated glycoprotein
      • Probstmeier R.
      • Fahrig T.
      • Spiess E.
      • Schachner M.
      Neural cell adhesion molecule
      • Probstmeier R.
      • Fahrig T.
      • Spiess E.
      • Schachner M.
      Platelet receptors
      • Moroi M.
      • Jung S.M.
      Promastigotes
      • Lira R.
      • Rosales-Encina J.L.
      • Arguello C.
      Staphylococcus aureus cell surface molecules
      • Rich R.L.
      • Deivanayagam C.C.
      • Owens R.T.
      • Carson M.
      • Hook A.
      • Moore D.
      • Symersky J.
      • Yang V.W.
      • Narayana S.V.
      • Hook M.
      S. aureus matrix-binding proteins
      • Hartford O.
      • McDevitt D.
      • Foster T.J.
      Streptococcus cricetus and Streptococcus rattus binding molecules
      • Liu T.
      • Gibbons R.J.
      • Hay D.I.
      Syndecan-1
      • Koda J.E.
      • Rapraeger A.
      • Bernfield M.
      Tenascin-C
      • Bachmann M.
      • Conscience J.F.
      • Probstmeier R.
      • Carbonetto S.
      • Schachner M.
      Uterine stromal cell CSPGs
      • Carson D.D.
      • Julian J.
      • Jacobs A.L.
      Vitronectin
      • Gebb C.
      • Hayman E.G.
      • Engvall E.
      • Ruoslahti E.
      von Willebrand factor
      • Pareti F.I.
      • Fujimura Y.
      • Dent J.A.
      • Holland L.Z.
      • Zimmerman T.S.
      • Ruggeri Z.M.
      If one considers that protein comprises roughly 20% of body mass (
      • Brozek J.
      • Grande F.
      • Anderson J.T.
      • Keys A.
      ) and that 30% or more of total protein is represented by collagen (
      • Lehninger A.L.
      Biochemistry.
      ,
      ), and if it is assumed that type I collagen comprises at least 90% of total collagen, it can be calculated that an adult of 70 kg may contain more than 1 × 1021 type I collagen monomers. Thus, the D-period repeat of type I collagen, with its host of numerous functional domains, is perhaps the most prevalent module of extracellular information in the body. Yet, despite the ubiquity and functional significance of type I collagen, unlike for other matrix molecules such as fibronectin and laminin, a map including all of its known functional domains and ligand-binding sites has been unavailable. Thus, here we have begun construction of a map of type I collagen with a view toward identifying relevant interactions and spatial relationships between type I collagen, its ligands, and disease-associated mutations.

      RESULTS AND DISCUSSION

      Distribution of Ligand-binding Sites and Functional Domains on Type I Collagen

      When the overall distributions of ligand-binding sites and functional domains on type I collagen are examined, three concentrations of sites, or potential hot spots of interaction, are apparent (Fig. 2, boxed regions 1, 2, and 3). These include a region near the N terminus, encompassing residues from about 80–200 (region 1), a more C-terminal region encompassing residues from about 680–830 (region 2), and another extending from about residue 920 to the C terminus (region 3). In general, more ligand-binding sites and functional domains map to the C-terminal half of the collagen molecule. Also, within the D-period most of the ligands interact with multiple sites on the fibril, which always map to two to three monomers, and for each ligand, there is never more than one binding site per monomer. These observations underscore the potential importance of intermonomer multivalency in ligand-fibril binding.

      Integrin-binding Sites

      Type I collagen is considered an important substrate for cell adhesion, and receptors reported to mediate cell-type I collagen interactions include the α1β1 and α2β1 integrins (
      • Knight C.G.
      • Morton L.F.
      • Onley D.J.
      • Peachey A.R.
      • Messent A.J.
      • Smethurst P.A.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      ,
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ), cell surface HSPGs (
      • Koda J.E.
      • Bernfield M.
      ), and the discoidin domain receptors 1 and 2 (DDR1 and DDR2) (
      • Shrivastava A.
      • Radziejewski C.
      • Campbell E.
      • Kovac L.
      • McGlynn M.
      • Ryan T.E.
      • Davis S.
      • Goldfarb M.P.
      • Glass D.J.
      • Lemke G.
      • Yancopoulos G.D.
      ,
      • Schlessinger J.
      ,
      • Vogel W.
      • Gish G.D.
      • Alves F.
      • Pawson T.
      ). Of the integrin receptors, a number of interactive sites have been mapped. The site at residue positions 435–438 was proposed to mediate platelet α2β1 integrin-collagen binding (
      • Staatz W.D.
      • Walsh J.J.
      • Pexton T.
      • Santoro S.A.
      ,
      • Staatz W.D.
      • Fok K.F.
      • Zutter M.M.
      • Adams S.P.
      • Rodriguez B.A.
      • Santoro S.A.
      ); however, this was not subsequently confirmed (
      • Knight C.G.
      • Morton L.F.
      • Onley D.J.
      • Peachey A.R.
      • Messent A.J.
      • Smethurst P.A.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      ,
      • Grab B.
      • Miles A.J.
      • Furcht L.T.
      • Fields G.B.
      ). Another site at residues 567–569 was implicated in RGD-dependent cell adhesion (
      • Dedhar S.
      • Ruoslahti E.
      • Pierschbacher M.D.
      ) but was later shown relevant only for denatured collagen (
      • Gullberg D.
      • Gehlsen K.R.
      • Turner D.C.
      • Ahlen K.
      • Zijenah L.S.
      • Barnes M.J.
      • Rubin K.
      ). Major α1β1 and α2β1integrin-binding sites for native type I collagen have subsequently been localized to 10 regions approximately including residues 84–187, 488–541, and 724–829 by ultrastructural mapping of binding sites between integrin receptors and procollagen monomers (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ). Putative integrin binding sequences were identified using two approaches. Thus, triple helical peptides were synthesized to generate a family of overlapping sequences spanning the cyanogen bromide peptide 3 of the α1(I) chain, and the GFPGER sequence at residues 502–508 was shown to be responsible for binding the von Willebrand Factor A-like domains (A-domains) of the α2β1 and α1β1 receptors as well as for mediating platelet- and HT1080 cell-type I collagen attachment (
      • Knight C.G.
      • Morton L.F.
      • Peachey A.R.
      • Tuckwell D.S.
      • Farndale R.W.
      • Barnes M.J.
      ). GER or GER-like motifs were found to be present in all of the integrin-binding domains; thus, collagenous peptides were synthesized: GLPGERGRP (most N-terminal site) (Fig. 2, region 1), GFPGERGVQ (central site), and GASGERGPP (most C-terminal site) (Fig. 2, region 2) (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ); all peptides inhibited recombinant α2- and α1-A-domain binding to type I collagen, although the latter was less active. Thus, the α2β1 and α1β1 receptors may share up to three ligand-binding sites on the type I collagen fibril.

      Potential Interplay between Integrin- and Matrix-binding Sites

      Considering only some of the more recently mapped integrin-binding sites, several potentially significant relationships between these regions and other interactive sites become apparent from the collagen D-period map. For example, four of the integrin-binding sites (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ,
      • Emsley J.
      • Knight C.G.
      • Farndale R.W.
      • Barnes M.J.
      • Liddington R.C.
      ) and the fibronectin-binding site (
      • Kleinman H.K.
      • McGoodwin E.B.
      ,
      • Kleinman H.
      • McGoodwin E.B.
      • Martin G.R.
      • Klebe R.J.
      • Fietzek P.P.
      • Woolley D.E.
      ,
      • Dzamba B.J.
      • Wu H.
      • Jaenisch R.
      • Peters D.M.
      ), although far apart in terms of primary collagen sequence, are quite close neighbors in the fibril (Fig. 2, region 2 and integrin-binding sites on adjacent monomer). This may relate to the observation that, at least for certain cell types, one mode of attachment to native type I collagen is fibronectin-dependent and is mediated by these integrin receptors (
      • Schoen R.C.
      • Bentley K.L.
      • Klebe R.J.
      ). Moreover, the site of binding of the decorin core protein (
      • Keene D.R.
      • San Antonio J.D.
      • Mayne R.
      • McQuillan D.J.
      • Sarris G.
      • Santoro S.A.
      • Iozzo R.V.
      ), which has been shown to interact with fibronectin (
      • Winnemoller M.
      • Schmidt G.
      • Kresse H.
      ), is also adjacent to several of the integrin-binding sites (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ) (Fig. 2, regions 2 and 3). This arrangement of sites could thus permit the formation of complexes of any of the integrin receptors, fibronectin, and the decorin core protein, which potentially could play a role in cell-type I collagen interactions. Engagement of type I collagen by cell surface PGs can also potentially modulate the activities of integrin-collagen interactions in a positive or negative manner (
      • Brennan M.J.
      • Oldberg A.
      • Hayman E.G.
      • Ruoslathi E.
      ). Of note is that the site proposed to mediate heparin/HSPG binding to type I collagen, at position 87–90 (
      • San Antonio J.D.
      • Lander A.D.
      • Karnovsky M.J.
      • Slayter H.S.
      ,
      • Sweeney S.M.
      • Guy C.A.
      • Fields G.B.
      • San Antonio J.D.
      ), overlaps with one of the α1β1-binding sites, is a near neighbor of the α2β1-binding site on the same monomer (Fig.2, region 1), and is a potential neighbor of several of the other α1β1- and α2β1-binding sites as they approximately align with each other on a vertical axis within the D-period (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ,
      • Emsley J.
      • Knight C.G.
      • Farndale R.W.
      • Barnes M.J.
      • Liddington R.C.
      ). Thus, potentially, binding of HSPGs or of heparin to type I collagen could modulate integrin-collagen interactions directly (through competition for occupancy of overlapping binding sites) or indirectly (through steric hindrance of binding sites by the GAG chains). Likewise, one of the binding sites for the guanidine HCl-extracted decorin PG (
      • Yu L.
      • Cummings C.
      • Sheehan J.K.
      • Kadler K.E.
      • Holmes D.F.
      • Chapman J.A.
      ), which presumably interacts with the collagen fibril in a GAG chain-dependent, core protein-independent manner (
      • Ramamurthy P.
      • Hocking A.M.
      • McQuillan D.J.
      ), overlaps with two of the integrin-binding sites on the collagen fibril (Fig. 2, region 1), and the other site of guanidine HCl-extracted decorin binding is a near neighbor of these integrin-binding sites.

      PG-binding Sites

      Interactions of type I collagen with PGs are also believed to be important in the regulation of the assembly and arrangement of collagen (
      • Keene D.R.
      • San Antonio J.D.
      • Mayne R.
      • McQuillan D.J.
      • Sarris G.
      • Santoro S.A.
      • Iozzo R.V.
      ,
      • Vogel K.G.
      • Paulsson M.
      • Heinegard D.
      ,
      • Danielson K.G.
      • Baribault H.
      • Holmes D.F.
      • Graham H.
      • Kadler K.E.
      • Iozzo R.V.
      ,
      • Scott J.E.
      • Haigh M.
      ,
      • Scott J.E.
      ). Regions of PG binding to fibrils have been deduced using GAG hydrolase treatments that degrade specific classes of GAG chains followed by staining of the GAG chains remaining associated with the fibril and visualization of binding regions using electron microscopy. In addition, the DSPG decorin has been localized on collagen fibrils relative to the D-period using antibodies against its core protein (
      • Fleischmajer R.
      • Fisher L.W.
      • MacDonald E.D.
      • Jacobs Jr., L.
      • Perlish J.S.
      • Termine J.D.
      ). It has been found that KSPGs and DSPGs/decorin bind to specific broad regions of the fibril (
      • Scott J.E.
      • Haigh M.
      ).

      Potential Interplay between PGs and Other Collagen-associated Molecules

      The region of KSPG binding, to the “c bands” of the fibril (
      • Scott J.E.
      ), overlaps with the phosphophoryn-binding site (
      • Dahl T.
      • Sabsay B.
      • Veis A.
      ), the major C-terminal intermolecular cross-link site (
      • Piez K.A.
      • Reddi A.H.
      Extracellular Matrix Biochemistry.
      ), and notably with the overall region of collagen glycation and with all of the described glycation sites at amino acid residues 434 on the α1(I) chain and residues 453, 479, and 924 on the α2(I) chain (
      • Reiser K.M.
      • Amigable M.A.
      • Last J.A.
      ). This latter observation may indicate a possible relationship between KSPG occupancy on the fibril and collagen modification seen in diabetes and aging. The other main region of KSPG binding, to the “a bands” of the fibril, overlaps with the major N-terminal intermolecular cross-link site (
      • Piez K.A.
      • Reddi A.H.
      Extracellular Matrix Biochemistry.
      ), the heparin/HSPG-binding site (
      • San Antonio J.D.
      • Lander A.D.
      • Karnovsky M.J.
      • Slayter H.S.
      ), a sequence mediating α2β1 integrin binding to denatured collagen (
      • Dedhar S.
      • Ruoslahti E.
      • Pierschbacher M.D.
      ,
      • Gullberg D.
      • Gehlsen K.R.
      • Turner D.C.
      • Ahlen K.
      • Zijenah L.S.
      • Barnes M.J.
      • Rubin K.
      ), the sites for vertebrate collagenase cleavage (
      • Wu H.
      • Byrne M.H.
      • Stacey A.
      • Goldring M.B.
      • Birkhead J.R.
      • Jaenisch R.
      • Krane S.M.
      ), and fibronectin binding (
      • Kleinman H.K.
      • McGoodwin E.B.
      ,
      • Kleinman H.
      • McGoodwin E.B.
      • Martin G.R.
      • Klebe R.J.
      • Fietzek P.P.
      • Woolley D.E.
      ,
      • Dzamba B.J.
      • Wu H.
      • Jaenisch R.
      • Peters D.M.
      ). Of potential significance to fibril assembly is the fact that this KSPG-binding region (
      • Scott J.E.
      ) also overlaps with all of the domains shown to be important for collagen fibrillogenesis (Fig. 2, regions 2 and 3) (
      • Prockop D.J.
      • Fertala A.
      ). At present, the positions of various types of KSPGs associated with type I collagen, e.g. lumican and keratocan, have not been immunolocalized on the collagen fibril, but if the different KSPGs are found to occupy unique zones on the type I collagen fibril, the relationships between KSPG types and the various functionally important sites on the fibril may be quite distinctive. The region of DSPG/CSPG/decorin binding, to the “d” and e bands“ of the fibril (
      • Scott J.E.
      ), overlaps with several integrin-binding sites (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ), both of the sites for guanidine HCl-extracted decorin binding (
      • Yu L.
      • Cummings C.
      • Sheehan J.K.
      • Kadler K.E.
      • Holmes D.F.
      • Chapman J.A.
      ), and one of the cartilage oligomeric matrix protein (COMP)-binding regions (
      • Rosenberg K.
      • Olsson H.
      • Morgelin M.
      • Heinegard D.
      ).

      Other Potential Interactions

      The binding site of phosphophoryn (
      • Traub W.
      • Jodaikin A.
      • Arad T.
      • Veis A.
      • Sabsay B.
      ,
      • Dahl T.
      • Sabsay B.
      • Veis A.
      ), a protein proposed to mediate mineralization of dentin, overlaps with a binding site for COMP (Fig. 2, region 2) (
      • Rosenberg K.
      • Olsson H.
      • Morgelin M.
      • Heinegard D.
      ), a thrombospondin family member found in cartilage and tendon (
      • Smith R.K.
      • Zunino L.
      • Webbon P.M.
      • Heinegard D.
      ), although the potential functional implications of this observation are unclear. These sites also map closely to overlapping sequences that mediate fibronectin binding (
      • Kleinman H.K.
      • McGoodwin E.B.
      ,
      • Kleinman H.
      • McGoodwin E.B.
      • Martin G.R.
      • Klebe R.J.
      • Fietzek P.P.
      • Woolley D.E.
      ,
      • Dzamba B.J.
      • Wu H.
      • Jaenisch R.
      • Peters D.M.
      ) and fibrillogenesis (
      • Prockop D.J.
      • Fertala A.
      ), cleavage by vertebrate collagenase (
      • Piez K.A.
      • Reddi A.H.
      Extracellular Matrix Biochemistry.
      ), and inhibition of collagen fibrillogenesis (Fig. 2, region 2) (
      • Prockop D.J.
      • Fertala A.
      ), implying the potential for fibronectin to function in the control of collagen assembly or degradation. The binding site for thrombospondin-1 has also been mapped to site(s) near the termini of tropocollagen (collagen monomers), but since the polarity of the tropocollagen used in that study was not established, it cannot be ascertained whether the thrombospondin-1- and the C-terminal COMP-binding sites on type I collagen are overlapping (
      • Galvin N.J.
      • Vance P.M.
      • Dixit V.M.
      • Fink B.
      • Frazier W.A.
      ).
      It should be noted that the map presents at least two discrepancies. Thus, although the fibril region shown to interact with the decorin PG in native fibrils overlaps with two regions of denatured decorin binding (e.g. GAG chain-mediated) (
      • Yu L.
      • Cummings C.
      • Sheehan J.K.
      • Kadler K.E.
      • Holmes D.F.
      • Chapman J.A.
      ,
      • Fleischmajer R.
      • Fisher L.W.
      • MacDonald E.D.
      • Jacobs Jr., L.
      • Perlish J.S.
      • Termine J.D.
      ), these sites do not overlap with the region on the monomer shown to bind the decorin core protein (
      • Keene D.R.
      • San Antonio J.D.
      • Mayne R.
      • McQuillan D.J.
      • Sarris G.
      • Santoro S.A.
      • Iozzo R.V.
      ). Likewise, the sites of phosphophoryn interaction on the fibril versus the monomer are nonoverlapping (
      • Traub W.
      • Jodaikin A.
      • Arad T.
      • Veis A.
      • Sabsay B.
      ,
      • Dahl T.
      • Sabsay B.
      • Veis A.
      ). Potential reasons for these discrepancies are discussed elsewhere (
      • Keene D.R.
      • San Antonio J.D.
      • Mayne R.
      • McQuillan D.J.
      • Sarris G.
      • Santoro S.A.
      • Iozzo R.V.
      ,
      • Dahl T.
      • Sabsay B.
      • Veis A.
      ).

      Distributions of Mutations Associated with Connective Tissue Diseases

      OI Mutations

      OI is a debilitating brittle bone disorder that affects roughly one in 10,000 individuals (
      • Byers P.H.
      • Wallis G.A.
      • Willing M.C.
      ). OI is commonly subdivided into four clinical types: type 1 (mild), 2 (lethal), 3 (severe), and 4 (moderately severe) (
      • Byers P.H.
      • Wallis G.A.
      • Willing M.C.
      ). While the majority of individuals with OI carry mutations in either of the genes coding for type I collagen, there are a number of OI patients and families where the collagen I defect is not the cause of the OI phenotype (
      • Glorieux F.H.
      • Rauch F.
      • Plotkin H.
      • Ward L.
      • Travers R.
      • Roughley P.
      • Lalic L.
      • Glorieux D.F.
      • Fassier F.
      • Bishop N.J.
      ,
      • Wallis G.A.
      • Sykes B.
      • Byers P.H.
      • Mathew C.G.
      • Viljoen D.
      • Beighton P.
      ,
      • Aitchison K.
      • Ogilvie D.
      • Honeyman M.
      • Thompson E.
      • Sykes B.
      ). Type I collagen defects can be divided into two main groups: quantitative and qualitative defects. The quantitative defect is typically a COL1A1-null allele, i.e. only 50% of the normal α1(I) chain is expressed, and it is commonly caused by introduction of a premature termination codon by a nonsense mutation or out-of-frame deletion/insertion. The phenotypic consequences of these mutations are virtually location-independent and result in an OI 1 phenotype (
      • Willing M.C.
      • Deschenes S.P.
      • Slayton R.L.
      • Roberts E.J.
      ). The qualitative defect usually involves a substitution of a bulkier amino acid for the obligatory glycine of the repeating Gly-X-Y (see Fig. 2legend) sequence of the collagen triple helix or an addition or deletion of amino acids by in-frame insertions or deletions, respectively (
      • Prockop D.J.
      • Kivirikko K.I.
      ,
      • Byers P.H.
      • Wallis G.A.
      • Willing M.C.
      ). The genotype-phenotype comparison of the qualitative defects has proven to be difficult. Based on the fact that mutations closer to the N terminus appear to exhibit milder phenotypes the gradient theory has been put forth. Thus, since collagen polymerizes from the C- to N-terminal direction, it is proposed that the N-terminal mutations would less significantly disrupt monomer assembly (
      • Prockop D.J.
      • Kivirikko K.I.
      ,
      • Byers P.H.
      • Wallis G.A.
      • Willing M.C.
      ). On the other hand, OI mutations on the α2(I) chain distribute to five alternating regions associated with either nonlethal or lethal phenotypes, supporting a regional model of OI pathophysiology (
      • Wang Q.
      • Orrison B.M.
      • Marini J.D.
      ). It has also been observed that substitution of glycine by amino acids such as aspartate, valine, and arginine is more deleterious than substitutions by serine, cysteine, and alanine (
      • Mooney S.D.
      • Huang C.C.
      • Kollman P.A.
      • Klein T.E.
      ,
      • Beck K.
      • Chan V.C.
      • Shenoy N.
      • Kirkpatrick A.
      • Ramshaw J.A.
      • Brodsky B.
      ) because of their more disruptive effects on triple helix stability. However, whereas these generalizations mostly seem to hold true, there are a number of exceptions.
      To better understand OI pathogenesis, one must define the mechanisms underlying OI phenotypic diversity and how OI arises from mutations in genes coding for type I collagen versus, for example, from those for other proteins that might function in concert with type I collagen. It also would be worthwhile to determine whether phenotypes associated with mutations arise through effects on type I collagen structure and/or its intermolecular interactions. To begin addressing such issues, we have examined the distributions of the various classes of OI mutations not only on the collagen monomer, as has been done by others previously, but also on the D-period map, which is a novel way to look at this information.
      On the monomer, it was observed that OI 1 mutations predominate (∼70%) in the N-terminal half, whereas OI 2 mutations slightly predominate (∼60%) in the C-terminal half as reported previously (
      • Prockop D.J.
      • Kivirikko K.I.
      ,
      • Byers P.H.
      • Wallis G.A.
      • Willing M.C.
      ). However, whereas OI 3 mutations were represented roughly equally in both the N- and C-terminal halves of the molecule, those associated with OI 4 were slightly more abundant (∼60%) in the N-terminal portion. When mutation positions were examined relative to the D-period structure, it was observed that in contrast to OI 2 mutations, which are more or less evenly distributed throughout, those associated with OI 3 and 4 show apparently nonrandom distributions, being excluded from several regions within the overlap zone, yet appearing to be more or less randomly distributed in the gap zone, which has not previously been reported (Fig. 3). Also of note is that 18 OI 3 and 4 mutations localize to the α2 chain between residues 226–301 where no OI 2 mutations have been reported.
      Figure thumbnail gr3
      Figure 3Distribution of mutations associated with OIand other diseases in the type I collagen D-period. Mutation positions are indicated by lines placed adjacent to amino acid positions at which mutations have been reported. A, OI 1 mutations (dashed lines indicate stop mutations);B, OI 2 mutations; C, OI 3 mutations;D, OI 4 mutations; E, mutations associated with non-OI disease phenotypes.

      Non-OI Mutations

      The map reveals another novel observation related to the distributions of mutations associated with osteoporosis (OPO) or osteopenia (OPA). Of these, an OPO and an OPA mutation localize within 2.3 nm of each other near the N terminus of the type I collagen monomer, and two of the other OPO mutations, although falling 225 residues apart from each other on the triple helix, localize to an area of only about 4 nm2 on adjacent monomers of the D-period (Fig. 2). When the relationship between these four mutations relative to adjacent D-periods is examined, all of them fall within an area of ∼74 nm2, or ∼20% of the D-period, in areas above or to the right of the gap or “hole zone,” a D-period structure proposed to be important in hydroxyapatite crystal growth during tissue mineralization (
      • Weiner S.
      • Traub W.
      ). These OPO or OPA mutations may just be very mild forms of OI since OI is characterized by reduced mineral deposition and bone fragility, or it may be that they primarily affect the function of type I collagen sequences involved in mineral deposition. Besides the OPO and OPA mutations, there are four other mutations reported in the triple helical region of the collagen I to be associated with mild, non-OI phenotypes in humans including cervical artery dissections, Marfan syndrome, connective tissue weakness, and Ehlers-Danlos syndrome. It is notable that these four non-OI mutations cluster together with the OPO/OPA mutations in two zones of the fibril, totaling less than 25% of the area of the D-period (Fig.3E). Potentially, certain D-period regions may be less critical to fibril function and thus may tolerate the presence of such mutations. Alternatively, these mutations may affect one or more classes of type I collagen intermolecular interactions, which collectively result in a spectrum of relatively mild connective tissue phenotypes distinct from OI.

      Dentinogenesis Imperfecta/OI Mutations

      Dentinogenesis imperfecta is characterized by hypomineralization of the teeth resulting in translucent and discolored teeth accompanied by increased fractures and chipping (
      • Lund A.M.
      • Jensen B.L.
      • Nielsen L.A.
      • Skovby F.
      ). Five substitution mutations have been shown to result in phenotypes that include OI and dentinogenesis imperfecta in the same individuals; three of them map to the gap zone, and the other two fall in the overlap zone but quite close to the overlap zone borders (Fig. 2), which largely excludes these mutations from the central region of the overlap zone.

      Distribution of Regions Lacking Reported Mutations

      A previous study has examined the distribution of about 80 OI mutations on the type I collagen monomer (
      • Scott J.E.
      • Tenni R.
      ). It was calculated that if the mutations were randomly distributed along the triple helix, the existence of any regions containing up to four glycines and devoid of mutations would be improbable (
      • Scott J.E.
      • Tenni R.
      ). In fact, such regions were observed, and it was proposed that they could potentially be so critical to collagen function that mutations that may occur in such areas are embryonically lethal and thus fail to appear in the human population (
      • Scott J.E.
      • Tenni R.
      ). On the other hand, the presence of regions lacking mutations could reflect their lack of importance to collagen function as mutations in such areas may not result in any disease phenotype. Thus, we examined our collagen map that contains the locations of almost 300 mutations to identify regions of the triple helix that lack mutations on both the α1(I) and α2(I) chains and limited our search to those that encompass four or more glycines. We identified 11 such areas (Fig.4A), many of which overlap with hot spots for ligand interactions (Fig. 4A,regions 2 and 6–11). Interestingly, each of the domains proposed to mediate α1β1 and α2β1 integrin binding (
      • Xu Y.
      • Gurusiddappa S.
      • Rich R.L.
      • Owens R.T.
      • Keene D.R.
      • Mayne R.
      • Hook A.
      • Hook M.
      ) map closely to one or two silent regions, although only region 10 overlaps with a putative integrin binding sequence containing the GER motif. On the other hand, a 28-residue stretch (Fig. 4A, region 1) that lacks reported mutations also has no proposed functions or sites for ligand interaction. Of potential significance is that many of these regions are approximately of similar sizes and are not distributed randomly in that for three of the monomers in the D-period they appear in blocks of two to four that occupy half or less of the D-period width. Moreover, when their arrangement is examined on monomers placed close to one another, as they may appear in the collagen fibril, most of the sites show slight to complete overlap with other sites on neighboring monomers (Fig. 4B). We thus speculate that the sites silent for mutations in type I collagen are critical for collagen function and may act cooperatively to promote collagen fibrillogenesis or to facilitate interactions with type I collagen-associated molecules, such as type V collagen (
      • Linsenmayer T.F.
      • Fitch J.M.
      • Birk D.E.
      ), proposed to be important for fibril assembly or function.
      Figure thumbnail gr4
      Figure 4Distribution of regions of the type I collagen D-period that lack reported mutations. A, regions lacking mutations and that encompass four or more glycines are indicated by hatched white boxes and numbered consecutively from the N to C terminus. B, same information as shown in A, but intermonomer distances have been reduced to better represent the close intermolecular distance of monomers in the type I collagen fibril and to illustrate the significant overlap between many of the regions lacking mutations in the type I collagen fibril.

      Summary

      Our D-period map of type I collagen has revealed three hot spots for ligand interactions, that most binding partners may interact with up to two or three monomers across the fibril, and the existence of a number of potentially significant relationships between ligand-binding sites and functional domains. Moreover, it was found that for several types of connective tissue disorders mutation positions within the D-period appeared to correlate with disease phenotype. In particular, OI 3 and OI 4 mutations appear to be more sparsely distributed in the overlap zone than in the gap zone, and most strikingly, the eight mutations associated with non-OI phenotypes are all restricted to within or near the gap zone. To best evaluate mutation distribution patterns in the fibril, however, would involve analysis of the positions of many more mutations than are currently available as well as a consideration of the frequency of each mutation in the human population. Our observations underscore the importance of examining type I collagen function at multiple levels, in two dimensions as we have done and eventually in three dimensions, once the structure of the type I collagen fibril has been fully elucidated. Such endeavors will help to reveal how type I collagen interacts with its cellular, extracellular matrix, cytokine, and other binding partners to generate and maintain tissue form and function.

      Acknowledgments

      We thank Andrew S. Likens for considerable efforts in performing the artwork in the design and construction of the type I collagen map and the other figures. We thank Dr. Karl Kadler of the University of Manchester for comments on decorin-collagen interactions and Dr. Magnus Hook and co-workers of Texas A&M University for sharing data on integrin-binding sites before publication.

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