Elsevier

Methods in Enzymology

Volume 499, 2011, Pages 167-182
Methods in Enzymology

Chapter nine - Hsp47 as a Collagen-Specific Molecular Chaperone

https://doi.org/10.1016/B978-0-12-386471-0.00009-2Get rights and content

Abstract

Heat shock protein (HSP) 47 is a 47 kDa collagen-binding glycoprotein localized in the endoplasmic reticulum (ER). It belongs to the serpin family and contains a serpin loop, although it does not have serine protease inhibitory activity. The induction of Hsp47 by heat shock is regulated by a heat shock element in its promoter region, while the constitutive and tissue-specific expression of Hsp47 correlates with that of collagen and is regulated via enhancer elements located in the promoter and intron regions. Hsp47 transiently binds to procollagen in the ER and dissociates in the cis-Golgi or ER-Golgi intermediate compartment region (ERGIC). Gene ablation studies indicated that Hsp47 is essential for embryonic development and the maturation of several types of collagen. The requirement for Hsp47 in collagen maturation may reflect its ability to inhibit collagen aggregation by binding procollagen in the ER and facilitate triple helix formation. In Hsp47-deficient cells, misfolded procollagen aggregates in the ER are degraded by the autophagy–lysosome pathway but not through the ubiquitin proteasome pathway. Hsp47 may be a therapeutic target for collagen-related disorders such as fibrosis, which feature abnormal accumulations of collagen and increased expression of Hsp47. This is supported by mouse models of fibrosis in which knockdown of Hsp47 clearly decreased the accumulation of collagen in fibrotic tissues and prevented the promotion of fibrosis. On the other hand, mutations in Hsp47 cause collagen-related genetic diseases such as osteogenesis imperfecta. Thus, Hsp47 is an indispensible molecular chaperone specific for collagen that is important in several major human diseases.

Introduction

In response to a range of stresses such as heat shock, cells respond by inducing a group of proteins called heat shock proteins (HSPs) (Richter et al., 2010). Most HSPs, many of which are well conserved from bacteria to mammals, function as molecular chaperones which facilitate the correct folding of nascent polypeptides into their native conformations and prevent aggregate formation by misfolded proteins in response to various stress conditions (Bukau et al., 2006). Thus, molecular chaperones maintain protein homeostasis under normal and stress conditions.

Molecular chaperones are located in various organelles, although many kinds are located at a high concentration in the endoplasmic reticulum (ER), a major organelle in which approximately 30% of total cellular proteins are synthesized, particularly secretory and membrane proteins (Ghaemmaghami et al., 2003, Huh et al., 2003). In addition to molecular chaperones such as glucose-regulated protein 78 (BiP), productive folding of nascent polypeptides in the ER also requires various enzymes involved in N-glycosylation and disulfide bond formation between cysteine residues (Anelli and Sitia, 2008). Only correctly folded proteins are allowed to be transported from the ER to their final destinations. In response to various stresses in the ER (“ER stress”), once protein folding is impaired and misfolded proteins are accumulated in the ER, which triggers the ER stress, various ER-resident molecular chaperones are induced. Molecular chaperones thus induced by ER stress are involved in refolding of misfolded proteins as well as degradation of potentially toxic misfolded or aggregated proteins, the mechanism of which is called “ER quality control.”

Hsp47 is an ER-resident stress protein with a unique character as a molecular chaperone that specifically binds to procollagen. Interestingly, Hsp47 is the only stress protein in the ER induced by heat shock; the other ER stress proteins are induced by ER stress. While other molecular chaperones have broad substrate specificity, Hsp47 specifically binds to procollagens in the ER and in addition, Hsp47 has a characteristic expression pattern in cells and tissues that correlates closely with collagen expression (Nagata, 1996, Nagata, 1998). Hsp47 belongs to the serpin (serine protease inhibitor) superfamily but does not have inhibitory activity for serine proteases (Nagata, 2003a).

In this chapter, we review the molecular function of Hsp47 in collagen biogenesis, the importance of Hsp47 in mouse development, and the clinical importance of Hsp47 in various collagen-related diseases including fibrosis.

Section snippets

Hsp47 as a Collagen-Binding Protein in the ER

Hsp47 was first identified in chick embryos as a 47 kDa collagen-binding protein resident in the ER, with a basic isoelectric point (pI = 9.0; Nagata and Yamada, 1986). Hsp47 was shown to be a homologue of colligin in mice (Kurkinen et al., 1984) and gp46 in rats (Cates et al., 1987). The molecular function of Hsp47 is one of the most well characterized among serpin family proteins located in the ER (Ragg, 2007).

The expression of Hsp47 is dramatically enhanced by heat shock (Nagata and Yamada,

Interaction and Recognition of Collagen by Hsp47

In vitro analysis using the surface plasmon resonance revealed that recombinant mouse Hsp47 can bind to collagen types I–V with dissociation constants of 10 6–10 7 M (Natsume et al., 1994). Estimates of dissociation and association rate constants suggested a rapid association and dissociation, which is likely to contribute to the transient nature of the interaction between Hsp47 and procollagen in the secretory pathway (Nakai et al., 1992). Based on pulse-chase experiments, Hsp47 can rapidly

A Phenotype and Abnormal Collagen Maturation in Hsp47 Knockout Mice

To further study the biological function of Hsp47 in vivo, hsp47 gene was disrupted in mice using the homologous recombination technique (Nagai et al., 2000). Mice lacking hsp47 did not survive beyond 11.5 days postcoitus (dpc), indicating that Hsp47 is essential for mouse development. At 10.5 dpc, the embryos of hsp47−/− mice were still viable but were about one size third of those of the wild type and showed developmental retardation and cardiac hypertrophy.

Collagen fibril accumulation in the

Possible Roles of Hsp47 in Procollagen Maturation in the ER

After incorporating the possible function of Hsp47, we can draw the following brief sketch of procollagen maturation in the ER. Newly synthesized polypeptides of procollagen, which are translated as procollagen chains, are cotranslationally inserted into the ER (Lamande and Bateman, 1999, Nagata, 1996). In the case of type I collagen, two α1 chains and one α2 chain form a trimer linked by disulfide bonds at the C-terminal domain. Intramolecular folding of polypeptides inserted into the ER is

Hsp47 Null Cells: A Tool for Studying the Fate of Misfolded Collagen

In hsp47−/− fibroblasts, triple helix formation of procollagen was totally impaired and its secretion was markedly inhibited or delayed, leading to the accumulation of procollagen in the ER (Ishida et al., 2006). In general, cells are equipped with two strategies to dispose of intracellular misfolded proteins: the autophagy–lysosome and ubiquitin–proteasome systems (Vembar and Brodsky, 2008). We investigated which of these pathways contributed to the disposal of accumulated procollagen in the

Regulation of Hsp47 Expression and Its Clinical Importance

As described above, Hsp47 expression is induced by heat shock but not by ER stresses. The constitutive expression of Hsp47 correlates tightly with collagen expression in various tissues and cell types. Indeed, Hsp47 expression is observed in collagen-producing cells such as fibroblasts, cartilages, and adipocytes, but not in collagen nonproducing cells such as neurocytes and myelocytes (Nagata, 2003b). Hsp47 possesses a Heat Shock Element (HSE) in its promoter region, and its induction by heat

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