The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 μM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.

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The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells

Blood ◽

10.1182/blood.v77.6.1171.1171 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1171-1180 ◽

Cited By ~ 15

Author(s):

CA Luhrs

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Binding Protein ◽

Binding Property ◽

Affinity Matrix ◽

Kb Cells ◽

Folate Binding Protein ◽

Binding Function ◽

High Mannose ◽

Endoglycosidase H

Abstract The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.

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Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

Bioscience Reports ◽

10.1023/a:1013234231960 ◽

2001 ◽

Vol 21 (3) ◽

pp. 305-313 ◽

Cited By ~ 11

Author(s):

Jan Holm ◽

Steen Ingemann Hansen ◽

Mimi Høier-Madsen

Keyword(s):

Ligand Binding ◽

Binding Protein ◽

Tryptophan Fluorescence ◽

Exchange Chromatography ◽

Cow’S Milk ◽

Conformation Change ◽

Ionic Charge ◽

Folate Binding Protein ◽

Cow's Milk ◽

Isoelectric Points

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.

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The role of glycosylation in the biosynthesis and acquisition of ligand- binding activity of the folate-binding protein in cultured KB cells

Blood ◽

10.1182/blood.v77.6.1171.bloodjournal7761171 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1171-1180

Author(s):

CA Luhrs

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Binding Protein ◽

Binding Property ◽

Affinity Matrix ◽

Kb Cells ◽

Folate Binding Protein ◽

Binding Function ◽

High Mannose ◽

Endoglycosidase H

The biosynthesis, processing, and ligand-binding function of the membrane-associated and soluble forms of the folate-binding protein (FBP) in KB cells, a cultured human cell line, were studied using pulse- chase labeling with [35S] methionine. The intermediary and mature forms of the protein were isolated by immunoprecipitation and affinity chromatography and analyzed by sodium dodecyl sulfate electrophoresis and autoradiography. The earliest species identified had an Mr of 32 Kd and disappeared over 5 hours concomitant with the appearance of a 38-Kd cellular FBP. As the 38-Kd species disappeared, a 40-Kd form appeared in the medium. When tunicamycin was added to the culture medium to inhibit core glycosylation, a 26-Kd aglycosylated species and minor 28- Kd and 30-Kd forms appeared. Endoglycosidase H, which cleaves high mannose but not complex oligosaccharides, reduced the 32-Kd species to 26-Kd but the enzyme had no effect on the 38-Kd form, indicating that this species is complex glycosylated. Monensin, which blocks complex glycosylation, also inhibited synthesis of the 38-Kd species. Although both the 32-Kd and 38-Kd forms had ligand-binding sites (as demonstrated by binding to a folate-Sepharose matrix), the 26-Kd aglycosylated species, labeled in the presence of tunicamycin, lacked similar binding sites because it did not bind to the affinity matrix. In contrast, the aglycosylated 26-Kd form, which was obtained by treatment of the 32-Kd species with endoglycosidase H, did bind to the folate affinity matrix, indicating that it retained ligand-binding function. Thus, the high mannose oligosaccharide moiety is not required for the folate-binding property of the FBP, but its addition to the polypeptide chain precedes a later step that is necessary for the mature protein to have ligand-binding function.

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Mechanical Strength and Inhibition of theStaphylococcus aureusCollagen-Binding Protein Cna

mBio ◽

10.1128/mbio.01529-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 38

Author(s):

Philippe Herman-Bausier ◽

Claire Valotteau ◽

Giampiero Pietrocola ◽

Simonetta Rindi ◽

David Alsteens ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Monoclonal Antibodies ◽

Ligand Binding ◽

Mechanical Strength ◽

Mechanical Response ◽

Binding Protein ◽

Adhesion Proteins ◽

High Affinity ◽

Living Bacteria

ABSTRACTThe bacterial pathogenStaphylococcus aureusexpresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bondsin vivois very strong (~1.2 nN), consistent with the high-affinity “collagen hug” mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation.IMPORTANCECna is a collagen (Cn)-binding protein fromStaphylococcus aureusthat is involved in pathogenesis. Currently, we know little about the functional role of the repetitive B region of the protein. Here, we unravel the mechanical strength of Cna in living bacteria. We show that single Cna-Cn bonds are very strong, reflecting high-affinity binding by the collagen hug mechanism. We discovered that the B region behaves as a nanospring capable of sustaining high forces. This unanticipated mechanical response, not previously described for any staphylococcal adhesin, favors a model in which the B region has a mechanical function that is essential for strong ligand binding. Finally, we assess the antiadhesion activity of monoclonal antibodies against Cna, suggesting that they could be used to inhibitS. aureusadhesion.

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Role of high-affinity folate-binding protein in the plasma distribution of tetrahydrofolate in pigs

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1996.270.1.r105 ◽

1996 ◽

Vol 270 (1) ◽

pp. R105-R110 ◽

Cited By ~ 1

Author(s):

K. Sasaki ◽

M. Natsuhori ◽

M. Shimoda ◽

Y. Saima ◽

E. Kokue

Keyword(s):

Protein Binding ◽

Binding Capacity ◽

Binding Protein ◽

Folate Binding Protein ◽

High Affinity ◽

Plasma Distribution ◽

The Stability ◽

Plasma Ultrafiltrate

Stability and protein-binding properties of tetrahydrofolate (THF) in pig plasma were studied in vitro. THF in plasma was stable for more than 120 min when it existed in a bound form, whereas THF both in plasma ultrafiltrate and in plasma ultrafiltrate plus porcine albumin was degraded rapidly and disappeared soon after its addition. These results suggest that high-affinity folate-binding protein (HFBP) is related to the stability of THF. THF-protein binding kinetic analysis showed that porcine plasma had HFBP and low-affinity binding protein (albumin) for THF. Dissociation constant and maximal binding capacity of HFBP were calculated to be 0.4 and 70 nM, respectively, indicating that > 98% of endogenous plasma THF existed in bound form with HFBP. Porcine albumin was not essentially a protein that binds and protects endogenous THF from degradation. We conclude that most endogenous THF binds to HFBP and only the unbound form of THF is rapidly degraded in pig plasma. HFBP protects THF from degradation and allows THF to exist stably in pig plasma. In addition, HFBP may govern the species specificity of plasma folate distribution in pigs.

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Effect of Hydrogen Ion Concentration and Buffer Composition on Ligand Binding Characteristics and Polymerization of Cow's Milk Folate Binding Protein

Bioscience Reports ◽

10.1023/a:1015528606487 ◽

2001 ◽

Vol 21 (6) ◽

pp. 745-753 ◽

Cited By ~ 6

Author(s):

Jan Holm ◽

Steen Ingemann Hansen

Keyword(s):

Ligand Binding ◽

Binding Protein ◽

Hydrogen Ion ◽

Ion Concentration ◽

Cow’S Milk ◽

Folate Binding Protein ◽

Hydrogen Ion Concentration ◽

Binding Characteristics ◽

Cow's Milk ◽

Buffer Composition

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10−6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).

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Ligand-induced conformation change in folate-binding protein

Biochemical Journal ◽

10.1042/bj2920921 ◽

1993 ◽

Vol 292 (3) ◽

pp. 921-925 ◽

Cited By ~ 32

Author(s):

N C Kaarsholm ◽

A M Kolstrup ◽

S E Danielsen ◽

J Holm ◽

S I Hansen

Keyword(s):

Fluorescence Spectroscopy ◽

Ligand Binding ◽

Binding Protein ◽

Simultaneous Detection ◽

Tryptophan Fluorescence ◽

Random Coil ◽

Folate Binding Protein ◽

Beta Strand ◽

Process Detection ◽

Induced Aggregation

C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure. Folate binding to FBP results in significant changes in the c.d. spectrum. Analysis of the spectrum shows a 10% decrease in antiparallel beta-strand as a result of ligand binding. Folate binding also leads to strong quenching of FBP tryptophan fluorescence. The magnitude of the quench is proportional to ligand binding. The guanidinium chloride-induced unfolding of FBP is shown to be a multistate process. Detection by c.d. and fluorescence spectroscopy lead to non-identical transitions. Modelling studies are consistent with the existence of a stable folding intermediate. Ligand binding to FBP increases the apparent folding stability of the molecule. Simultaneous detection by c.d. and fluorescence indicate that the apparent increased folding stability is derived from ligand-induced aggregation of FBP.

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