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. 2009 Feb 27;284(9):5539-45.
doi: 10.1074/jbc.M806369200. Epub 2008 Dec 22.

Differences in chain usage and cross-linking specificities of cartilage type V/XI collagen isoforms with age and tissue

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Differences in chain usage and cross-linking specificities of cartilage type V/XI collagen isoforms with age and tissue

Jiann-Jiu Wu et al. J Biol Chem. .

Abstract

Collagen type V/XI is a minor but essential component of collagen fibrils in vertebrates. We here report on age- and tissue-related variations in isoform usage in cartilages. With maturation of articular cartilage, the alpha1(V) chain progressively replaced the alpha2(XI) chain. A mix of the molecular isoforms, alpha1(XI)alpha1(V)alpha3(XI) and alpha1(XI)alpha2(XI)alpha3(XI), best explained this finding. A prominence of alpha1(V) chains is therefore characteristic and a potential biomarker of mature mammalian articular cartilage. Analysis of cross-linked peptides showed that the alpha1(V) chains were primarily cross-linked to alpha1(XI) chains in the tissue and hence an integral component of the V/XI polymer. From nucleus pulposus of the intervertebral disc (in which the bulk collagen monomer is type II as in articular cartilage), type V/XI collagen consisted of a mix of five genetically distinct chains, alpha1(XI), alpha2(XI), alpha3(XI), alpha1(V), and alpha2(V). These presumably were derived from several different molecular isoforms, including alpha1(XI)alpha2(XI)alpha3(XI), (alpha1(XI))(2)alpha2(V), and others. Meniscal fibrocartilage shows yet another V/XI phenotype. The findings support and extend the concept that the clade B subfamily of COL5 and COL11 gene products should be considered members of the same collagen subfamily, from which, in combination with clade A gene products (COL2A1 or COL5A2), a range of molecular isoforms has evolved into tissue-dependent usage. We propose an evolving role for collagen V/XI isoforms as an adaptable polymeric template of fibril macro-architecture.

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Figures

FIGURE 1.
FIGURE 1.
SDS-PAGE of pepsin-extracted types II and XI collagens from 4-year bovine articular cartilage. Samples were run on SDS-6% polyacrylamide gel and stained with Coomassie Brilliant Blue R250. Lane 1, type II collagen (0.8 m NaCl fraction). Lane 2, type XI collagen (1.2 m NaCl fraction). Further analysis by reverse-phase HPLC showed that α1(V) chains co-migrated with α1(XI) (Figs. 2 and 3).
FIGURE 2.
FIGURE 2.
Reverse-phase HPLC of α1(V), α1(XI), α2(XI), and α3(XI) collagen chains extracted by pepsin from bovine articular cartilage at different developmental ages. A, fetal cartilage; B, 18-month cartilage; C, 4-year cartilage. Purified type XI collagen preparations were denatured and chromatographed on a C4 column to resolve chain components (see “Experimental Procedures” for details).
FIGURE 3.
FIGURE 3.
Reverse-phase HPLC resolution of the α-chains of pepsin-extracted type XI collagen from adult (4-year) bovine articular cartilage. The [3H]NaBH4-treated type XI collagen molecules were denatured and chromatographed on a C4 column to resolve chain components. Aliquots of collected fractions (100 μl) were assayed for tritium activity and by SDS-6% PAGE (inset).
FIGURE 4.
FIGURE 4.
Reverse-phase HPLC fractionation of tryptic peptides prepared from α1(XI), α2(XI), and α1(V) chains of 4-year bovine articular cartilage. Samples (1 mg) were eluted from a C8 column (Brownlee Aquapore R-300; 25 cm × 4.6 mm) with a linear gradient (0–35%) of solvent B in solvent A over 70 min at a flow rate of 1 ml/min. Solvent A was 0.1% trifluoroacetic acid (v/v) in water, and solvent B was 0.085% trifluoroacetic acid (v/v) in acetonitrile:n-propyl alcohol (3:1, v/v). Also shown are the amino-terminal sequences of the isolated tritiated peptides. P*, 4-hydroxyproline; K*, hydroxylysine; and X, cross-linking hydroxylysine residue.
FIGURE 5.
FIGURE 5.
DEAE-HPLC fractionation of native type V/XI collagen molecules from adult bovine articular cartilage. Molecular forms of type XI/V collagen were partially resolved on a Bio-Gel DEAE-5-PW column under non-denaturing conditions. Fractions (fxn) were desalted and analyzed by SDS-6% PAGE.
FIGURE 6.
FIGURE 6.
Reverse-phase HPLC and SDS-PAGE two-dimensional resolution of chain components of the collagen V/XI fraction from pepsin-digested calf nucleus pulposus. The sample (1 mg) was denatured and resolved on a C4 column monitoring for UV absorbance (lower panel). Fractions were analyzed by SDS-6% PAGE (upper panel). The conditions were as in Fig. 4. Individual collagen chains were identified by in-gel trypsin digestion and mass spectrometry.
FIGURE 7.
FIGURE 7.
SDS-PAGE/Western blot analysis of type V/XI collagen chains resolved by HPLC from nucleus pulposus. Aliquots of pooled fractions in Fig. 6 were resolved by SDS-6% PAGE and transblotted to polyvinylidene difluoride membrane. The blots were reacted with polyclonal antisera specific to the N-telopeptide cross-linking sequences of α1(XI) (A) and α2(XI) (B) to detect α-chains with these telopeptides still attached through cross-linking.
FIGURE 8.
FIGURE 8.
SDS-PAGE and Western blot analysis of type V/XI collagen chains resolved by HPLC from calf meniscus. The conditions are as in Fig. 6. Upper panel, Coomassie Brilliant Blue-stained gel. Lower panel, Western blot using a polyclonal antiserum specific against α1(XI) N-telopeptide cross-linking domain. fxn, fraction.

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