Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking

Abstract

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

FIGURE 1.

SDS-PAGE patterns of cross-linking collagens. Collagen types I (lane 2), III (lane 4), and IV (lane 6) each displayed a banding pattern distinct from the other two. RFUVA causes collagen type I (lane 3) α and β chains to almost disappear, γ chains to increase slightly in intensity, and protein staining at the base of the gel well to increase. A similar pattern of band disappearance is seen also with type III collagen (lane 5) and type IV collagen (lane 7), with increased staining at the top of the gel (base of the sample well). Lane 1, molecular mass standards. Gels were stained with Coomassie Blue.

FIGURE 2.

SDS-PAGE patterns of cross-linking core proteins. RFUVA caused the monomer band of each PG to virtually disappear. For keratocan, lumican, and mimecan, RFUVA + PG did not cause a higher M_r band to form, instead forming new higher M_r polymers of a wide range of sizes from 100/150 kDa to the base of the gel well (lanes 3, 5, and 7). For decorin, RFUVA caused an entirely new band to form of approximately twice the size of the monomer band (lane 9). Gels were stained with Coomassie Blue.

FIGURE 3.

Interaction of core proteins keratocan, lumican, and mimecan with type I collagen. Presence of keratocan or lumican prevented RFUVA from causing diminished staining of α chains and β chains of collagen (lanes 5 and 7). In contrast, mimecan did not interfere with collagen cross-linking, thus allowing α chains and β chains to be cross-linked to form high M_r polymers near γ chains in size and toward the top range of the gel, and staining intensity increased sharply at the base of the sample well (lane 9). Gels were stained with Coomassie Blue.

FIGURE 4.

Interaction of core protein decorin with type I collagen. In the presence of RFUVA (lane 7), decorin did not interfere with collagen cross-linking, as evidenced by the virtual disappearance of α and β chains, the increased density of γ chains, and the same increased density of staining in the upper region of the gel as in the absence of decorin (lane 3). Gels were stained with Coomassie Blue.

FIGURE 5.

Western blot of proteoglycan core protein interaction with type I collagen induced by RFUVA. Western blot is shown. A, the presence of keratocan prevented RFUVA from causing collagen type I to undergo cross-linking (lane 4 versus lane 2). The intensities of α and β chains (lane 4) were almost the same as the corresponding bands in lane 1. B, lumican inhibited collagen cross-linking (lane 4 versus lane 2). C, mimecan did not inhibit RFUVA-induced collagen cross-linking (lane 2 versus lane 4), whereas mimecan formed a new band near γ chains and in the top range of the gel (lane 8 versus lane 7). D, decorin did not inhibit RFUVA-induced collagen cross-linking (lane 2 versus lane 4), whereas decorin formed a new band near γ chains (lane 8 versus lane 7). Decorin dimers and oligomers induced by RFUVA were observed (lane 7).

FIGURE 6.

Effect of keratocan or lumican on RFUVA cross-linking of collagen in the simultaneous presence of both mimecan and decorin. Western blot is shown. A, in the simultaneous presence of both mimecan and decorin, collagen cross-linking in response to RFUVA is not inhibited, and both mimecan and decorin each undergo their respective patterns of cross-linking. B, in contrast, in the presence of keratocan, as well as mimecan and decorin, collagen I cross-linking by RFUVA was partly inhibited (lane 3 versus lane 2). In addition, RFUVA-induced cross-linking of both decorin and mimecan in the presence or absence of collagen I are inhibited (lane 4 versus lane 5 and lane 6 versus lane 7). C, similarly, in the presence of lumican, collagen I cross-linking by RFUVA was partly inhibited (lane 3 versus lane 2). In addition, RFUVA-induced cross-linking of both decorin and mimecan in the presence or absence of collagen I is inhibited (lane 4 versus lane 5 and lane 6 versus lane 7).

FIGURE 7.

Analysis to detect possible interaction of KS and collagen type I, using mass spectrometry. Peaks at m/z 462.0 (A–D) correspond to the characteristic KS-monosulfated disaccharide released by keratanase II from gel slices (area from 100 kDa down to the bottom of the SDS gel). There are no peaks at m/z 462.0 (E–H) observed with gel slices in the area from 150 kDa up to the base of the gel sample well.

FIGURE 8.

Analysis to detect possible interaction of CS and collagen type I, using mass spectrometry. Peaks at m/z 458.0 (A–D) correspond to the characteristic CS monosulfated disaccharide (4S/6S) released by chondroitinase ABC from gel slices (area from 100 kDa down to the bottom of the SDS gel). There are no peaks at m/z 458.0 (E–H) observed with gel slices in the area from 150 kDa to the base of the gel sample well.

FIGURE 9.

Effect of RFUVA on collagen type I cross-linking in whole cornea ex vivo versus collagen type I in solution. A, patterns of collagen type I from whole cornea treated by RFUVA. Lanes 1 and 2, GHCl extraction; lanes 3 and 4, pepsin extraction. B, patterns of collagen type I from whole cornea treated by RFUVA and then digested with CNBr. Lanes 1 and 2, GHCl extraction; lanes 3 and 4, pepsin extraction. C, patterns of electrophoretic migration of purified collagen type I in solution, treated by RFUVA, and digested with CNBr. All peptide domains of collagen type I participate in cross-linking to form a range of high M_r molecules. A and B, Western blot; C, Coomassie Blue staining.

FIGURE 10.

Patterns of cross-linking collagens and PG core proteins in whole cornea ex vivo. Western blot is shown. A, interaction of keratocan and collagen type I from corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5), N-glycanase (NGase) (lanes 6 and 7), and keratanase II (KSase) (lanes 8 and 9). B, interaction of lumican and collagen type I from corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5), N-glycanase (lanes 6 and 7), and keratanase II (lanes 8 and 9). C, interaction of mimecan and collagen type I from corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5), N-glycanase (lanes 6 and 7), and keratanase II (lanes 8 and 9). D, interaction of decorin and collagen type I from corneas (with or without RFUVA treatment) treated by CNBr (lanes 4 and 5), O-glycanase (OGase) (lanes 6 and 7), and chondroitinase ABC (ABCase) (lanes 8 and 9). All four PG core proteins in the cornea show some molecules that co-migrate in the same region as collagen γ chains (arrowheads).