Cross-Linking and the Molecular Packing of Corneal Collagen

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    Cross-Linking and the Molecular Packing of Corneal Collagen

    Mitsuo Yamauchi,*,1 Gloria S. Chandler,* Hideki Tanzawa, and Elton P. Katz*CB# 7455 Dental Research Center, University of North Carolina, Chapel Hill, North Carolina 27599-7455; Chiba

    University Medical School, Chiba, Japan; and University of Connecticut Health Center,Farmington, Connecticut 06032

    Received January 4, 1996

    We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as afunction of age. The major iminium, reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL)and dehydro- histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; how-ever, the latter persisted in mature animals at a level of 0.30.4 moles/mole of collagen. A nonreduciblecross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a majormature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similarto skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum valuewith time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filamentsthat are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linkingwould facilitate the special collagen swelling property required for corneal transparency. 1996 Academic Press,Inc.

    The transparency of cornea is thought to be due to the unique properties of its collagenous matrix(1). The collagen is present in uniformly sized heterotypic fibrils comprised of type I and type Vmolecules (2). The molecular packing in these fibrils have a number of special aspects. Like skinthey have an axial periodicity of 65 nm compared to 67 nm that is found in tendon or bone (3). Themolecules are also packed laterally much more loosely than any other type I collagen fibril, having,for example a most frequent intermolecular distance of 1.6 nm (4) compared to about 1.4 nm oftendon or skin (5).We have been determining the stoichiometry and stereochemistry of the naturally occurring

    cross-linking reactions that originate at the COOH and NH2 terminal ends of the molecules (6).These studies indicate that collagen fibrils have more than one packing modality. In ligament, boneand dentin fibrils, each molecule is surrounded by six nearest molecules that are offset by 1 to 4multiples of the axial periodicity (D); however, in skin the collagen molecules appear to beorganized into lamellae of near register (OD) molecules (see figure 1) (7,8).The aim of this study was to quantitatively characterize the collagen cross-linking of bovine

    cornea as a function of age as a step towards understanding the structure of the corneal collagenfibrils. As cross-linking is also a major determinant of fibrillar swelling, this data gives an insightinto the origins of the special hydration properties of cornea fibrils. Our results are the first todemonstrate a histidine-involved nonreducible cross-link, histidinohydroxylysinonorleucine(HHL), as a major stable cross-link in cornea and also to characterize the quantitative changes ofthe major corneal collagen cross-links as a function of age.

    METHODS AND MATERIALSCorneas were excised from eyes of various aged pre- and postnatal bovine animals and stored at 90C. The corneas were

    divided into five age groups; fetal (n 4 4), 12 yr (n 4 5), 56 yr (n 4 4), 78 yr (n 4 4) and older than 8 yr (n 4 1).Samples for analysis were pulverized to fine powder under liquid nitrogen by a Spex Freezer Mill (Spex Inc., Edison, NJ),washed with cold 0.015 M N-trismethyl-2-aminoethanesulfonic acid (TES) buffer, pH 7.4 and distilled water, and lyoph-ilized. Approximately 3 mg of each sample was suspended in 0.15 M TES buffer, pH 7.4, and reduced with standardized

    1 To whom correspondence should be addressed. Fax: (919)966-1231.


    3110006-291X/96 $18.00Copyright 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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    NaB3H4. The measurement of specific activity of NaB3H4 and the reduction with NaB3H4 were performed according to themethod described previously (9). The specific activity of the NaB3H4 used was 12.7 X 107 D.P.M./mmole. The reducedsamples were hydrolyzed with 6N HCl in vacuo, after flushing with N2, for 22 hrs at 105C. The hydrolysates were driedby a speed vacuum (Savant Instruments Inc.), dissolved in distilled water and filtered. An aliquot of each hydrolysate wassubjected to amino acid analysis to determine hydroxyproline. Then the hydrolysates with known amounts of hydroxy-proline were analyzed for cross-links on a Varian HPLC 5500 fitted with an ion-exchange column (AA911, Interaction) (9).This was linked to an on-line fluorescence flow monitor (Shimadzu Instrument Co.) and a liquid scintillation flow monitor(Flo-one Beta, Radiomatic Instrument). Using this system, reducible cross-links (iminium cross-links) and nonreducible,fluorescent cross-links (pyridinoline and deoxypyridinoline) were quantified (9). The histidine-based nonreducible cross-link, HHL, was identified and quantified by an amino acid analyzer using the HHL cross-link standard as describedpreviously (10, 11). The contents of HHL in some samples were confirmed by using different gradient systems on theanalyzer and/or by using the same gradient system after the bulk of amino acids in the hydrolysate was removed by thestandardized molecular sieve P2 column (11). The quantities of all cross-links analyzed were expressed in a moles per moleof collagen basis using a value of 300 residues of hydroxyproline per collagen molecule.

    RESULTSTypical chromatographic profiles of reducible and non-reducible cross-links of bovine corneal

    collagen obtained from three different age groups (fetal, 12 yr, 78 yr) are given in Figure 2. Themajor reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL, its reduced form,HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD, its reduced form, HHMD).Essentially no dehydro-dihydroxylysinonorleucine (deH-DHLNL) was detected in this tissue. Incontrast to mineralized tissue collagens (12), little free lysine/hydroxylysine aldehyde (5-amino-5-carboxypentanal/2-hydroxy-5-amino-5-carboxypentanal) was present in cornea in all age groups(

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    early maturational stage, persisted throughout life (0.30.4 mole/mole of collagen). The nonre-ducible fluorescent cross-links, pyridinoline and its lysyl analog (deoxypyridinoline), were virtu-ally absent from most corneas analyzed (

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    cross-link in skin collagen indicates that most of its molecules are packed in near register in oneof its dimensions, and are staggerred by 1D and 4D in the other directions (see figure 1) (7). TheHHL cross-link is absent not only from mineralized tissues but also from some major soft con-nective tissues such as tendon and ligament (11).The major HHL cross-linked peptide has been isolated from a tryptic digest of mature bovine

    cornea collagen and the amino acid composition indicated the molecular locus to be identical to thatof skin collagen (unpublished). The presence of HHL in corneal collagen fibrils at this molecularlocus suggests that, here too, the collagen molecules have a 0D packing motif. Such packing tendsto reduce the axial periodicity (8), and corneal collagen, like skin has a reduced axial periodicityof about 65 nm versus the 67 nm present in tendon or bone (3). Corneal fibrils also are comprisedof helicoidal filaments (3). The intermolecular interaction patterns of molecules packed in nearregister would introduce a chiral character to filaments (8).There are some differentiating aspects to corneal cross-linkung which have physiological im-

    plications. Significant amounts of HHL (0.2 mole./mole of collagen) exist in fetal animals and theHHL content reaches a steady level of 0.30.4 moles/mole soon after birth. Since type I collagencomprises about 85% of the collagen (2), this value indicates that only 1 HHL residue is formedper two type I collagen molecules. This is in contrast to skin where there is a steady build up ofHHL with time that approaches a 1 residue per type I collagen molecule in old bovines or humans(11). Moreover, there is a small overall intermolecular cross-linking in mature cornea fibrils,comprised almost entirely of HHL and deH-HHMD. The total cross-link content amounts to onlyless than one per collagen molecule, as compared to 3 per molecule in ligament and 2 per moleculein mature skin. This may be part of the explanation for the hyperswollen state of the corneal fibrilsas indicated by the anamously large value for the most frequent intermolecular distance. Anadditional factor is that corneal collagen fibrils are heterotypic, composed of type I and type Vmolecules, with the type V molecules being in the interior of the fibril. All else being the same, theinclusion of type V molecules into the fibrillar structure could localize HHL and deH-HHMDcross-linking, as illustrated in figure 1. This pattern of type V inclusion compartmentalizes the fibrilinto 3-molecular-stranded filaments, that are stabilized by both HHL and deH-HHMD linkages (8).

    ACKNOWLEDGMENTSThis work was supported by NIH Grants DE 10489, AR 37604 and NASA Grant NAGW-3946.

    FIG. 3. Changes of the major collagen cross-links with development, maturation and aging in bovine cornea. Thecontents of the cross-links are expressed in moles per mole of collagen based on a value of 300 residues of hydroxyprolineper collagen molecule (mean + SD, N 4 45 except for >8 year old group).



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