ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2

Abstract

B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46-47 °C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the K_d values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg²⁺ and Mn²⁺ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.

Keywords: ATPase; B-cell immunoglobulin binding protein; Glucose-regulated protein 78 kDa; endoplasmic reticulum chaperone; endoplasmic reticulum retention sequence KDEL; signal sequence.

Figure 1

Structure, cloning and visualisation of full‐length huBiP and variants. (A) Schematic illustration of recombinant human full‐length BiP and its variants. Signal sequence (orange; S), NBD (blue), SBD (violet), KDEL sequence (green; K). Amino acid numbering is based on information contained in Wang et al., 2017 and Zhang et al., 2010 1, 23. (B) SDS/PAGE of purified recombinant full‐length huBiP and variant BiP proteins. Proteins were expressed in BL21–CodonPlus (DE3)‐RIL cells and purified using metal chelate chromatography as described in the Materials and methods part. A 10% SDS‐polyacrylamide gel was used to separate the denatured protein. The Coomassie Brilliant Blue‐stained gel is presented. On each lane of the SDS gel, 15 μg of either purified full‐length BiP or variant proteins was loaded as indicated. (C) A representative image of native PAGE gel of purified full‐length BiP and variants. Fifteen microgram of purified full‐length BiP and variant proteins was loaded on each lane of a 6% polyacrylamide gel, and native PAGE was performed using Tris‐Glycine as running buffer. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue. (D) The quantification of the different protein species in panel C was carried out as described in the Materials and methods section. In diagram, the area under the absorption curve of the monomeric (mono.) BiP variant S⁻/K⁻ (last lane in panel C) was arbitrarily set to 1; dimeric (dim.) and oligomeric (oligo.) forms of each variant protein are presented as relative values.

Figure 2

Effect of nucleotides and cations on BiP. BiP S⁻/K⁻ (20 μg) in the presence of 2 mm of Mg²⁺ or Mn²⁺ or 1 mm of ATP or ADP and a combination of cations and nucleotides as indicated were incubated at 25 °C for 10 min and were loaded on a Bis‐Tris 6% gel, and native PAGE was performed using Tris‐Glycine as running buffer. After electrophoresis, the gel was then stained with Coomassie Brilliant Blue.

Figure 3

Biochemical characterisation of recombinant full‐length BiP and variant proteins thereof. ATPase activity of recombinant full‐length huBiP and three variant proteins was measured. ATPase activity assays were performed in a final volume of 160 μL containing 10 μg of protein with 30 mm HEPES‐KOH, pH 7.8, 150 mm NaCl, 20 μm ATP and 2 mm Mg²⁺. The proteins with the signal sequence had the highest activity compared to the other variants with no signal sequence. An unpaired Student's t‐test was performed, and significant differences were observed between full‐length huBiP and variant S⁻/K⁻ (P = 0.01), as well as between variants S⁻/K⁺ and S⁻/K⁻ (P = 0.03). Specific enzyme activity and respective standard deviation (SD) values were calculated using data from three individual batches for each recombinant protein variant.

Figure 4

Effects of divalent cations on the ATPase activity of BiP S⁺/K⁺ and S⁻/K^−. To test the influence of divalent cations on the BiP ATPase activity, increasing amounts of MgCl₂, MnCl₂ and CaCl₂ were added to the reaction mixture containing 20 μm ATP and 10 μg protein. ATPase assays were carried out in triplicate in a range from 0 to 10 mm of cations and representative results (mean of the values and SD) of such analyses are presented. MgCl₂ and MnCl₂ stimulated the ATPase activity of BiP with optimal concentrations of 100 and 25 μm, respectively. At higher concentrations of ≥ 1 mm, MgCl₂ still stimulated ATPase activity but to a slightly lesser extent, whereas MnCl₂ at concentration ≥ 1 mm failed to stimulate BiP ATPase activity and even inhibited it slightly. In contrast, calcium did not stimulate or inhibit the ATPase activity of BiP S⁺/K⁺. (A) BiP S⁺/K⁺ ATPase activity in the presence of Mg²⁺ and Mn²⁺ using a logarithmic scale for the cation presentation. The small inset shows ATPase activity of BiP S⁺/K⁺ at lower concentrations (0–100 μm) of Mg²⁺ and Mn²⁺ presenting the cation concentrations as a linear scale. (B) BiP S⁻/K⁻ ATPase activity in the presence of Mg²⁺ and Mn²⁺ with a logarithmic scale for the cations being used. The inset is the close‐up view of BiP S⁻/K⁻ ATPase activity at lower concentrations (0–100 μm) of Mg²⁺ and Mn²⁺ using a linear scale presentation of cation concentrations. (C) BiP S⁺/K⁺ ATPase activity in the presence of Ca²⁺ using a linear scale.

Figure 5

Biophysical characterisation of recombinant full‐length huBiP and variant proteins thereof. (A) Thermal stability analyses of recombinant full‐length huBiP and three variant proteins thereof were performed using DSF. The reaction mixtures contained 1 μm protein in 30 mm HEPES‐KOH, pH 7.8, 150 mm NaCl and 1× Sypro orange. The melting temperatures of the full length and variant proteins are in the range of 45.1–45.8 °C. The calculated T _m (e.g. the maximum of the first derivative of the raw data) is shown as a mean of triplicate experiments of three individual batches for each protein variants with respective SD error bars. An unpaired Student's t‐test was performed, and a significant difference was observed between S⁺/K⁺ and S⁻/K⁻ (P = 0.006). (B) Thermal denaturation profile of recombinant full‐length huBiP and variants with omitted N‐ or C‐terminal amino acids. The first peak (46.6 °C for S⁺/K⁺, 46.8 °C for S⁺/K⁻, 46.8 °C for S⁻/K⁺ and 46.6 °C for S⁻/K⁻) indicates the unfolding of the N terminus of these proteins, whereas the second peak (62.75 °C for S⁺/K⁺, 62.2 °C for S⁺/K⁻, 63 °C for S⁻/K⁺ and 62.4 °C for S⁻/K⁻) represents the unfolding of the C‐terminal domain of these proteins 8. The thermal denaturation profiles of full‐length huBiP and variant proteins suggest that the N‐ and C termini of these proteins are properly folded. Representative melting curves with calculated T _m are shown as a mean of three individual batches for each protein variant. Their low values of SDs of the melting temperatures suggest a very good reproducibility of the experiments (data not shown).

Figure 6

Nucleotide binding of huBiP variants. In DSF experiments presented here, 1 μm protein was incubated in 30 mm HEPES‐KOH, pH 7.8, 150 mm NaCl and 1× Sypro orange. The melting curve of the samples was measured from 25 to 95 °C with 1 °C increments increase in temperature. (A) The interaction of BiP S⁺/K⁺ and BiP S⁺/K⁻ with ATP was measured in the absence of divalent cations. The denaturation profiles of the proteins were studied in the presence of increasing amounts of ATP (0–40 mm). (B) The interaction of BiP S⁺/K⁺ and BiP S⁺/K⁻ with ADP was determined without divalent cations. Denaturation profiles of the proteins were studied in the presence of increasing amounts of ADP (0–10 mm). (C) The interaction of BiP S⁻/K⁻ and BiP S⁻/K⁺ with ATP was measured with no divalent cations present. The denaturation profiles of the proteins were studied in the presence of increasing amounts of ATP (0–40 mm). (D) The interaction of huBiP BiP S⁻/K⁻ and huBiP S⁻/K⁺ with ADP was determined without divalent cations. Denaturation profiles of the proteins were studied in the presence of increasing amounts of ADP (0–10 mm). In each diagram, the melting temperatures of the proteins as indicated (y‐axis linear scale) dependent on the nucleotide concentrations (x‐axis in logarithmic scale) are presented. The K _d values were calculated using graphpad prism software under the assumption of a simple cooperative model.

Figure 7

Effect of divalent cations on BiP S⁺/K⁺ and BiP S⁻/K⁻ nucleotide binding. In these DSF experiments, 1 μm protein was incubated in 30 mm HEPES‐KOH, pH 7.8, 150 mm NaCl and 1× Sypro orange. The melting curve of the samples was measured from 25 to 95 °C with 1 °C increments increase in temperature. (A) The interaction of BiP S⁺/K⁺ in the presence of increasing amounts of ATP (0–10 mm) was studied using 50 μm Mg²⁺ and 25 μm Mn² as indicated. (B) Interaction of BiP S⁺/K⁺ with increasing concentrations of ADP (0–10 mm) in the presence 50 μm Mg²⁺ and 25 μm Mn²⁺ was measured. (C) The binding of BiP S⁺/K⁺ to increasing amounts of ATP (0–10 mm) having 2 mm of divalent cations (Mg²⁺, Mn²⁺ and Ca^{2 +} as indicated) present was measured. (D) The interaction of BiP S⁺/K⁺ with increasing amounts of ADP (0–10 mm) in the presence of 2 mm of divalent cations (Mg²⁺, Mn²⁺ and Ca^{2 +}) was analysed. In each diagram, the melting temperatures of the proteins as indicated (y‐axis linear scale) dependent on the nucleotide concentrations (x‐axis in logarithmic scale) are presented. The K _d values were calculated using graphpad prism software under the assumption of a simple cooperative model.

Figure 8

BiP ATPase activities in the presence of low concentrations of divalent cations. ATPase activity of huBiP was measured in the presence of MgCl₂ (50 μm) and MnCl₂ (25 μm). Samples were prepared with a reaction volume of 160 μL containing 10 μg of protein, 30 mm HEPES‐KOH, pH 7.8, 150 mm NaCl, either 50 μm MgCl₂ or 25 μm MnCl₂ and increasing concentrations of ATP (0–3 mm). Experiments were repeated twice in each case but only one representative set of experiments is presented. (A) BiP S⁺/K⁺ ATPase activity in the presence of 50 μm Mg²⁺ and 25 μm Mn²⁺. (B) BiP S⁻/K⁻ ATPase activity in the presence of 50 μm of Mg²⁺ and 25 μm of Mn²⁺. The K _m and V _max values were calculated using the Michaelis‐Menten equation and graphpad prism software.

Figure 9

Model illustrating BiP‐ATP‐ and BiP‐ADP‐bound conformation. (A) Full‐length BiP tends to be in open conformation in the ATP‐bound state (indicated by thick blue arrow) in the presence of metal ions (Me²⁺), enabling easy exchange of ATP. (B) BiP S⁻/K⁻ tends to be in a closed conformation in the ADP‐bound state in the presence of Ca²⁺ and the binding of ADP is tighter.