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

ABSTRACT: A major collagen-binding heat-shock glycoprotein from L6 myoblasts, designated gp46, was purified by gelatin-agarose chromatography and ion-exchange chromatography. Purified gp46 was functionally active, as shown by its ability to rebind to gelatin-agarose, and was homogeneous as determined by SDS/polyacrylamide-gel electrophoresis. This is the first reported purification of myoblast gp46 in an active state. Triton X-100-soluble gp46 was found to bind preferentially to immobilized pepsin-treated type IV collagen compared with native type I collagen. gp46, reconstituted into phospholipid vesicles, retained collagen-binding activity. This activity could be destroyed by chemical modification with chloramine-T, but was decreased by only 20-30% following treatment with iodoacetamide or N-ethylmaleimide. Since gp46 is a heat-shock protein, we examined the possibility that it may confer protection on type I collagen from thermal denaturation at temperatures above its normal melting temperature of 41 degrees C. In the presence of gp46 liposomes the apparent melting temperature of type I collagen was marginally increased to 42 degrees C. This change was considered to be too small to be of physiological significance. We have therefore concluded that the role of gp46 in collagen metabolism is unlikely to be related to any thermal-stabilizing function.

SUBMITTER: Vaillancourt JP

PROVIDER: S-EPMC1149980 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Project description:Protein synthesis in rat L6 myoblasts is stimulated and protein breakdown inhibited in a co-ordinate manner by insulin-like growth factors (IGF) or insulin. For both processes, bovine IGF-1 was somewhat more potent than human IGF-1, which was effective at a tenth the concentration of insulin, rat IGF-2 or human IGF-2. A similar order of potency is noted when DNA synthesis or protein accumulation is monitored over a 24 h period, but between 20- and 50-fold higher concentrations of each growth factor are required than those needed to produce effects in the 4 h protein-synthesis or -breakdown measurements. Binding experiments with labelled human or bovine IGF-1 as ligand demonstrated competition at concentrations of IGF-2, especially human IGF-2, lower than that of either IGF-1 preparation. This pattern was much more pronounced when the radioligand was either human IGF-2 or rat IGF-2. Insulin competed 10-15% for the binding of labelled IGF-1, but not at all with labelled IGF-2. Ligand-receptor cross-linking experiments showed that labelled bovine IGF-1 bound approximately equally to the type 1 IGF receptor (Mr 130000 after reduction) and to the type 2 IGF receptor (Mr 270000 after reduction), and that unlabelled IGF-1 competed equally with radioligand binding to both receptors. On the other hand, rat IGF-2 competed more effectively for binding to the type-2 receptor, and insulin competed only for binding to the type-1 receptor. Further cross-linking experiments with rat IGF-2 as radioligand demonstrated binding only to the type-2 receptor and to proteins with Mr values after reduction of 230000 and 200000. This binding was prevented by high rat IGF-2 concentrations, less effectively by bovine IGF-1 and not at all by insulin. The apparently conflicting biological potencies and receptor binding of the different growth factors can be explained if all the biological actions are mediated via the type-1 IGF receptor, rather than through the abundant type-2 receptor.

Project description:When hookworm infective L3s infect their mammalian host, they undergo a temperature shift from that of the ambient environment to that of their endothermic host. Additionally, L3s living in the environment can be exposed to temperature extremes associated with weather fluctuations. The heat shock response (HSR) is a conserved response to heat shock and other stress that involves the expression of protective heat shock proteins (HSPs). The HSR is controlled by heat shock factor-1 (HSF-1), a conserved transcription factor that binds to a heat shock element in the promoter of HSPs, causing their expression. HSF-1 is negatively regulated in part by a HSF binding protein (HSB-1) that binds to and removes HSF-1 trimers bound to HSP gene promoters, resulting in attenuation of the HSR. Herein we describe an HSB-1 orthologue, Ac-HSB-1, from the hookworm Ancylostoma caninum. The Ac-hsb-1 cDNA encodes a 79 amino acid protein that is 71% identical to the Caenorhabditis elegans HSB-1, and is predicted to share the characteristic coiled-coil structural motif comprised of two interacting alpha helices. Recombinant Ac-HSB-1 immunoprecipitated Ce-HSF-1 expressed in mammalian cells that had been heat shocked for 1h at 42°C, but not from cells incubated at 37°C, indicating that HSB-1 only bound to the active DNA binding form of HSF-1. Expression of Ac-hsb-1 transcripts decreased following 1h of heat shock, but increased when L3s were incubated at 37°C for 1h. Activation of hookworm L3s induces a five-sixfold increase in Ac-hsb-1 expression that peaks at 12h, coincident with L3 feeding, but that subsequently decreases to two-threefold above control at 24h. Recombinant Ac-HSB-1 immunoprecipitates greater amounts of 70 and 40kDa proteins from extracts of activated L3s than from non-activated L3s. We propose that an increase in Ac-hsb-1 levels early in activation allows feeding to resume, but that a subsequent decrease in expression permits a HSR that protects non-developing L3s at host-like temperatures. Further investigations of the HSR will clarify the role of HSB-1 and HSF-1 in hookworm infection.

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

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