Purification and reconstitution of a collagen-binding heat-shock glycoprotein from L6 myoblasts

Vaillancourt, John P.; Cates, G A

doi:10.1042/bj2740793

Cited by 11 publications

(6 citation statements)

References 25 publications

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“…This observation is important, because procollagen rather than collagen is found in the ER. Previous work (Vaillancourt and Cates, 1991) has been limited to the qualitative binding of mature collagens with colligin. However, the binding of colligin to cleaved, mature collagen indicates that at least one of -the sites that colligin recognizes in procollagen lies in the central helical region.…”

Section: Discussionmentioning

confidence: 99%

“…After 4 h, wells were washed three times with buffer A, and bound colligin was quantified by an e.l.i.s.a. assay using anti-colligin antibody and rabbit anti-mouse IgG-horseradish peroxidase conjugate as described previously (Vaillancourt and Cates, 1991). The absorbance readings at 490 nm, after colour development with o-phenylenediamine reagent, were corrected for non-specific binding.…”

Section: Cross-linking and Immunoprecipitationmentioning

confidence: 99%

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Interaction of procollagen I and other collagens with colligin

Jain

Brickenden

Lorimer

et al. 1994

Biochemical Journal

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Colligin is a collagen-binding glycoprotein of molecular mass 46000 Da localized to the endoplasmic reticulum (ER) of diverse kinds of cells that produce collagen I. In order to help define its role in collagen biosynthesis and to study the interaction of colligin with procollagen I in detail, the binding characteristics of colligin purified from L6 myoblasts have been studied. A total of 3 mol were found to bind/mol of procollagen I, with a Kd of about 25 nM. Both pure and separated pro alpha 1(I) and procollagen alpha 2 (I) chains were able to compete with procollagen I for binding to colligin. However, colligin binds to pro alpha 2 (I) with higher affinity than to pro alpha 1 (I). To find if the binding activity of colligin was altered during purification, an assay to measure colligin binding to procollagen in crude myoblast cell extracts was developed. This procedure gave the same binding parameters as did the highly purified colligin. Among different collagen types, colligin was found to bind to collagen I and collagen IV, but not to collagen III. In order to examine whether glycosylation or phosphorylation of colligin were required for the binding of colligin to procollagen I and to obtain enough colligin for further studies, recombinant protein was produced in Escherichia coli. An immunoaffinity purification scheme was used to get virtually pure protein in milligram yields. Comparison of the recombinant colligin with that isolated from L6 myoblasts showed that both types existed in solution as monomers and dimers. In addition, both types of colligins showed identical properties with regard to their binding to procollagen I and the isolated pro alpha 1(I) and pro alpha 2(I) chains. Post-translational modifications of colligin were thus not essential for binding to procollagen I.

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Section: Discussionmentioning

confidence: 99%

Section: Cross-linking and Immunoprecipitationmentioning

confidence: 99%

Interaction of procollagen I and other collagens with colligin

Jain

Brickenden

Lorimer

et al. 1994

Biochemical Journal

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“…Purification of Chicken HSP47-The purification method was based upon previously published protocols for the purification of HSP47 (9,20) but has been modified to improve both the yield and purity of HSP47.…”

Section: Methodsmentioning

confidence: 99%

HSP47 Binds Cooperatively to Triple Helical Type I Collagen but Has Little Effect on the Thermal Stability or Rate of Refolding

Macdonald

Bächinger

2001

Journal of Biological Chemistry

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HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens. Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K half ) of 1.4 ؋ 10 ؊7 M, and a Hill coefficient of 4.3 was observed. Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli. Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker. Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed. Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix. Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.HSP47 (47-kDa heat shock protein) is a rough endoplasmic reticulum protein believed to function as a collagen-specific molecular chaperone (1-3). It was shown that HSP47 (also known as colligin, J6, gp46, and CB48) is co-expressed with collagens in cells such as fibroblasts and chondrocytes (4,5). In mouse embryo development, HSP47 is expressed mainly in mesoderm and mesoderm-derived tissues, and the expression correlates both temporally and spatially with that of type I and II collagen (6). HSP47 knockout mice show an embryonic lethal phenotype (7). HSP47 is isolated easily by affinity chromatography on gelatin-Sepharose (8, 9) and was later shown to interact also with triple helical collagen types I-V (10). It was found that all these collagens have a similar affinity for HSP47 with dissociation constants of about 10 Ϫ7 M (10). The interaction of HSP47 with both unfolded and folded procollagens is unusual and distinguishes HSP47 from other molecular chaperones.A number of potential functions for HSP47 during procollagen biosynthesis have been described. HSP47 binds to nascent procollagen chains and therefore may help prevent the premature association of procollagen chains and/or assist with the translocation of procollagen chains into the rough endoplasmic reticulum (11). HSP47 also interacts with procollagen chains in the rough endoplasmic reticulum when triple helix formation is inhibited by ␣,␣Ј-dipyridyl, an inhibitor of prolyl-4-hydroxylase (9). A potential role in vesicular trafficking was explored with Brefeldin A and monensin (9,12,13). In Brefeldin A-treated cells HSP47 remained associated with procollagen, whereas in cells treated with monensin no HSP47 binding was detected.Together this shows that HSP47 seems to release procollagen on entry to the Golgi. Procollagens are not secreted as individual molecules, but ...

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“…Since Hsp47 was reported to interact with collagen, we investigated the effect of bisecting GlcNAc of Hsp47 on their interaction. The interaction of Hsp47 and type 1 collagen was examined using a solid phase assay as reported (Vaillancourt and Cates, 1991). However, no obvious differences were detected in this preliminary experiment (data not shown).…”

Section: Discussionmentioning

confidence: 86%

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

Gao¹,

Miyoshi²,

Uozumi³

et al. 2005

Glycobiology

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The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.

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Purification and reconstitution of a collagen-binding heat-shock glycoprotein from L6 myoblasts

Cited by 11 publications

References 25 publications

Interaction of procollagen I and other collagens with colligin

Interaction of procollagen I and other collagens with colligin

HSP47 Binds Cooperatively to Triple Helical Type I Collagen but Has Little Effect on the Thermal Stability or Rate of Refolding

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

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