Intracellular trafficking and degradation of unassociated proα2 chains of collagen type I

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Abstract

Procollagen I is a trimer consisting of two proα1(I) chains and one proα 2(I) chain. In certain cases of mild osteogenesis imperfecta, abnormal proα1(I) chains are degraded very soon after synthesis. As a consequence, the cells produce excess proα2(I) chains, which cannot form trimers and are not secreted. The objective of this work was to determine the intracellular fate of unassociated proα2(I) chains. Mov13 mouse fibroblasts, which do not synthesize proα1(I) mRNA, but do produce proα2(I) mRNA, were incubated with radioactive amino acids using pulse-chase protocols, and proteins were analyzed by gel electrophoresis, autoradiography, and Western blotting. Mov13 cells produced proα2(I) chains that were degraded intracellularly within 30 min. Degradation was inhibited when cells were treated with brefeldin-A, which blocks transit from endoplasmic reticulum to Golgi. Fixed cells exposed to various immunofluorescence markers and imaged by confocal laser scanning microscopy showed that proα2(I) chains colocalized with Golgi and lysosome markers. Degradation was inhibited and chains were secreted when cells were treated with wortmannin, which blocks trafficking to lysosomes. These results demonstrate that unassociated proα2(I) chains leave the endoplasmic reticulum, transit the Golgi, and enter lysosomes where they are degraded.

Introduction

Collagens are a family of extracellular matrix proteins that are important in developmental processes and necessary components in the structure and proper functioning of tissues and organs such as heart, skin, and bone. Collagen assembles as fibers, filaments, or networks, either alone or in conjunction with other components of the extracellular matrix [1]. Procollagen I is a trimer composed of two proα1(I) chains and one proα2(I) chain whose sequences are encoded by two different genes. Heritable disorders in the biosynthesis, assembly, posttranslational modification, or fibrillogenesis of collagen can have profound clinical consequences [2].

Osteogenesis imperfecta (OI) is a family of heritable disorders caused, in many instances, by mutations that affect the primary structures of proα1(I) or proα2(I) chains; the disorders are characterized by symptoms that can be mild, severely deforming, or perinatal lethal. Mutations in the gene coding for proα1(I) chains may result in the production of mutant chains that cannot be incorporated into trimers. In other cases of mild OI, the mutant allele is silent. In both these cases, 50% less collagen type I is found in the extracellular matrix [2]. Aberrant proα1(I) chains produced by cultured skin fibroblasts from patients with lethal OI are degraded by a proteasome pathway associated with the endoplasmic reticulum (ER) [3], [4]. However, normal proα2 chains that are not incorporated into trimers are not degraded in this way nor are they secreted [3]. The objective of this study is to determine the intracellular fate of unassociated proα2(I) chains.

To address this issue, it is necessary to identify an appropriate model system. The heterozygous Mov13 mouse, which carries a proviral insert in the cis-regulatory region of the collagen 1a1 gene [5], produces 50% less proα1(I), has 50% less collagen I in the extracellular matrix, and exhibits characteristics of mild osteogenesis imperfecta including bone fragility [2], [6] and growth adaptations [7]. When heterozygous Mov13 mice are mated, the homozygous offspring die at midgestation [8]. Cells established from these embryos do not produce mRNA for proα1(I), but they do produce mRNA for proα2(I) chains [9]. It has been widely assumed that the chains are made and then rapidly degraded [8], but attempts to detect proα2(I) chains in these Mov13 cells have been unsuccessful. If this assumption is correct, then the Mov13 cells could serve as a model to study the fate of unassociated proα2(I) chains, such as are found in mild OI. Furthermore, this work might shed light on the mechanisms of protein surveillance and quality control as it relates to other oligomeric proteins in the secretory pathway [10].

Section snippets

Materials

Cell culture supplies including DME, MEM, penicillin/streptomycin (P/S) suspension (5000 U/ml penicillin G and 5000 μg/ml streptomycin sulfate in 0.85% saline), FBS, and PBS, were purchased from GIBCO BRL (Gaithersburg, MD). 14C-proline (248 mCi/mmol) or 14C-glycine (98.0 mCi/mmol) (Amersham, Buckinghamshire, UK) and trans 35S-Label™ (10 mCi/ml) (ICN, Costa Mesa, CA) were used to metabolically label proteins. The anti-procollagen type I antibody, SP 1.D8, which recognizes an epitope in the

Proα2(I) chains are made but not secreted by Mov13 cells

Proteins from Mov13 cells and media were harvested, resolved on SDS-PAGE, and electroeluted under standard conditions. After washing and blocking, as described above, the membrane was reacted with the primary antibody Sp1.D8. The secondary antibody–alkaline phosphatase conjugate was then applied and reacted with alkaline phosphatase substrate. Fig. 1 shows a 156-kDa protein present in the cells but not in the medium, demonstrating that Mov13 cells produce proα2(I) chains, but do not secrete

Mov 13 cells produce unassociated proα2(I) chains that are quickly degraded

This report presents the first direct evidence that proα2(I) chains are synthesized by Mov13 cells. There have been indirect indications that the chain is made but to date it has not been detected [8], [9], [24], [35], [36]. The reason why the chains were not observed until now is that cellular proteins from homozygous Mov13 are usually exposed to pepsin before analyzing them on SDS-PAGE [8], [24], [37]. This is a standard method for studying collagens, since the N- and C-propeptides are

Acknowledgements

This work was supported in part by grants from the Society of Cosmetic Chemists and Summer Research Fellowships from the New York Chapter of the Arthritis Foundation (to M.G.G.); and a Biomedical Science Grant from the Arthritis Foundation (R.S.B.). The Imaging Facility of Albert Einstein College of Medicine is supported in part by Cancer Center Support Grant P30-CA-13330 from the United States Public Health Service.

Some of the data presented here are contained in a dissertation submitted by

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