Elsevier

Matrix Biology

Volume 22, Issue 5, September 2003, Pages 449-453
Matrix Biology

Protein consequences of the Col2a1 C-propeptide mutation in the chondrodysplastic Dmm mouse

https://doi.org/10.1016/S0945-053X(03)00077-5Get rights and content

Abstract

The Disproportionate micromelia (Dmm) mouse has a three nucleotide deletion in Col2a1 in the region encoding the C-propeptide which results in the substitution of one amino acid, Asn, for two amino acids, Lys–Thr. Western blot and immunohistochemical analyses failed to detect type II collagen in the cartilage matrix of the homozygous mice and showed reduced levels in the matrix of heterozygous mice. Type II collagen chains localized intracellularly within the chondrocytes of homozygote and heterozygote tissues. These findings provide evidence that the expression of type II procollagen chains containing the defective C-propeptide results in an intracellular retention and faulty secretion of type II procollagen molecules. A complete absence of mature type II collagen from the homozygote cartilage and an insufficiency of type II collagen in the heterozygote cartilage explains the Dmm mouse phenotype. The integrity of the C-propeptide is thus crucial for the biosynthesis of normal type II collagen by chondrocytes.

Introduction

Fibrillar collagens reinforce most vertebrate tissues and multicellular animals in general. Type II collagen, the main structural protein of hyaline cartilage, is essential for skeletal development and the mechanical strength of mature articular cartilage. The molecule is a homotrimer of three identical α-helical polypeptide chains [pro-α1(II) chains], encoded by the gene Col2a1. The chains are synthesized as precursors with amino- and carboxy-terminal propeptide extensions. Mutations in COL2A1 cause human chondrodysplasias in the spondyloepiphyseal dysplasia spectrum ranging from lethal in utero to early onset osteoarthritis with a mild skeletal phenotype (Kuivaniemi et al., 1997).

The synthesis and extracellular secretion of fibril-forming collagens (types I, II, III and V/XI) are complex processes perhaps most extensively studied for types I and III collagens (reviewed in Kuivaniemi et al., 1997). It is assumed that types II and V/XI collagens follow a similar pathway of assembly. The C-propeptide is required for trimer formation. In type I collagen, mutations that alter the C-propeptide domain cause severe osteogenesis imperfecta (reviewed in Kuivaniemi et al., 1997). In type II collagen, only five dominant mutations within the COL2A1 C-propeptide domain have been reported; all cause forms of spondyloepiphyseal dysplasia (Fig. 1A). No recessive mutations of human COL2A1 are known.

The disproportionate micromelia (Dmm) mouse is the only known example of a homozygotic mutation in the type II collagen C-propeptide domain. Pace et al. (1997) identified a three nucleotide deletion in the Col2a1 C-propeptide coding sequence that results in a K206_T207delinsN (N substituted for KT, residue numbers beginning at the start of the C-propeptide). Homozygotes die at birth due to thoracic insufficiency. Heterozygotes appear normal at birth but develop a mild chondrodysplasia with skeletal dwarfing 1-week post-partum (Brown et al., 1981) and evidence of osteoarthritis beginning at 4 months (Seegmiller et al., 2001). Here we report analytical findings on the effect of this mutation on the expression and deposition of type II collagen in chondrocytes and cartilage matrix of heterozygous and homozygous Dmm mice. A complete lack of type II collagen in the cartilage matrix explains the severity of the observed homozygote phenotype.

Section snippets

Biochemical findings

Fig. 1B shows a Western blot comparing pepsin-extracted collagens from +/+, D/+ and D/D mouse rib cartilage. The monoclonal antibody (mAb) 1C10 shows the presence of α1(II) collagen chains in +/+ (lane 2) and +/− (lane 4) but not in D/D (lane 3) tissue. Type II collagen appeared, therefore, to be completely absent from the D/D fetal cartilage matrix, but was abundant in heterozygote cartilage as it was in the wild type. However, compared to +/+, there was approximately a 45% reduction in the

Discussion

The results show that type II collagen is synthesized and deposited in the cartilage matrix of the heterozygote, although at lower levels compared with wild-type tissue. This apparently is sufficient for the heterozygotes to grow normally in utero and appear skeletally normal at birth. The present findings are consistent with insufficient type II collagen being deposited in the extracellular matrix of cartilages during growth of the heterozygous animals and can explain the dwarfing phenotype

Collagen extraction, electrophoresis and Western blotting

Day-18 fetuses were obtained from heterozygote mouse matings and genotyped (Pace et al., 1997). Rib cartilage from wild-type control mice (+/+) and from mice homozygous (D/D) and heterozygous (D/+) for the Dmm mutation were weighed and extracted in a fixed volume per mouse (dissected rib cage) of 4 M guanidinium HCl, 50 mM Tris–HCl, pH 7.4, containing protease inhibitors for 24 h at 4 °C (Fernandes et al., 2001) to solubilize non-cross-linked type II collagen. Cross-linked type II collagen in

Uncited references

Ahmad et al., 1995, Unger et al., 2001

Acknowledgements

This study was supported by NIH grants AR 37318, HD 22657 and AR 47568. The authors thank Dr Laura C. Bridgewater, Brandon Bomsta and Peter Crane for genotyping the mice, Kae Ellingsen for help with formatting the manuscript and Tom Eykemans for help with the graphics.

References (15)

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