Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform

Abstract

Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1+ex2, ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1+ex2 mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.

Patient-Derived Skeletal Dysplasia Induced Pluripotent Stem Cells Display Abnormal Chondrogenic Marker Expression and Regulation byBMP2andTGFβ1

“…Color images available online at www.liebertpub.com/scd unprocessed form pro-a1(IIB), but not pro-a1(IIA), shown at *150 kDa and its cleaved form a1(II) at *120 kDa (Fig. 4C, Ch), as expected for mature differentiated chondrocytes [36,38,57]. Overall, immunofluorescence staining from micromasses and western blot data using micromass culture media for COL2 confirm at the protein level, the abnormal regulation of COL2A1 expression in TRPV4-iPSCs, seen by qPCR assays.…”

“…Our data indicate that the necessary up-regulation of this important marker of chondrogenesis is impaired in the TRPV4 mutant. Recently characterized homozygous Col2a1 + ex2 knock-in mice, where the IIA form is exclusively expressed, showed that cartilage tissue assembled into thin-banded fibrils [57], indicating that functional cartilage can be formed with only the IIA form of COL2 [62]. In addition, the relatively higher expression of IIA compared with IIB suggests that early, rather than late, stages of chondrogenesis are represented in our iPSC model during this time frame in culture.…”

Assessment of hyaline cartilage matrix composition using near infrared spectroscopy

“…Hydroxyproline which is one of the three major amino acids present in the collagen protein chain is frequently used to estimate the total collagen content in samples (Hosseininia et al, 2013;McAlinden et al, 2014). The amount of collagen in the cartilage powders averaged 72.9% weight/dry weight (w/dw) in adult articular cartilage, 64.1% w/dw in young articular cartilage and 41.5% w/dw in nasal cartilage.…”

The development of a mature collagen network in cartilage from human bone marrow stem cells in Transwell culture

“…Mutations in prolyl 3-hydroxylase isoenzymes, associated helper proteins that affect proline 3-hydroxylation [6–14] and mutations that alter the primary structure of the α1(II) collagen chains can disrupt fibrillogenesis and result in a disorganized cartilage extracellular matrix during development and growth [15–19]. Pro-986 has been shown to be highly 3-hydroxylated in α1(IIA/B) chains in mouse cartilage [20] and in the Swarm rat chondrosarcoma (RCS-LTC) cell line pN-α1(IIB) chains [21]. Pro-944 and Pro-707 are also 3-hydroxylated and positioned within 3 residues of the collagen D-period molecular stagger (234 amino acids) suggesting a supplementary role in collagen fibril assembly [5,21,22].…”

“…However, to what extent the fibrillar matrix is well developed, in terms of the post-translational biochemistry, assembly and cross-linking of type II collagen and its co-assembly with (types IX and XI collagens), has not been characterized. We have developed methods to screen for normal collagen co-polymeric assembly in order to monitor the quality of the matrix deposited by chondrocytes in culture and in cartilages affected by heritable or acquired disease states [20,33,34]. This study investigated the ability of human bone marrow stem cells undergoing chondrogenesis to deposit collagen in the neo-matrix and to assemble a type II collagen-based cross-linked network characteristic of cartilage.…”