27 research outputs found
Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous āblackā pigment gallstone formation in germfree Swiss Webster mice
āBlackā pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates (āhyperbilirubinbiliaā) from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.National Institutes of Health (U.S.) (Training Grant T32-OD10978-26)National Institutes of Health (U.S.) (Training Grant P30-ES002109)Kinship Foundation. Searle Scholars Progra
Bioinorganic Chemistry of the Human Host-Defense Protein Calprotectin
Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.The human innate immune system responds to bacterial and fungal pathogens by releasing the metal-chelating protein calprotectin (CP) at sites of infection and in the upper layers of the epidermis. CP is a Mn(II)- and Zn(ll)-binding protein. The work described in this thesis elucidates the metal-binding properties of CP, and correlates these properties with in vitro growth inhibition of bacteria and fungi. We report that the metal-binding properties of CP are modulated by Ca(ll), and we propose a working model in which CP responds to physiological Ca(Il)-ion gradients to become a potent Zn(ll)- and Mn(Il)-chelating agent in the extracellular space. Individual chapter summaries follow. Chapter 1: Bioinorganic Chemistry of the Host Pathogen Interaction. Transition metal ions are required for all forms of life. During the course of infection, pathogenic microorganisms must acquire transition metals from the host. Three metals of interest from this standpoint are iron, zinc, and manganese. This chapter describes bacterial metal-ion homeostasis machineries, and metal-requiring processes with a focus on Zn(II) and Mn(II). This chapter then highlights the S100 family of Ca(ll)-binding proteins and discuses the Zn(Il)-, Cu(ll)-, and Mn(Il)-binding properties of S100B, S100A12, S100A7, S10OA15, and S100A8/S100A9. Finally, an overview of the scope of this thesis is presented. Chapter 2: Calcium Ion Gradients Modulate the Zinc(Il) Affinity and Antibacterial Activity of Human Calprotectin. Calprotectin (CP) is a human neutrophil protein that is produced and released by neutrophils at sites of infection, where it prevents the growth of microorganisms by sequestering bioavailable zinc(II) and manganese(II). In this chapter, we present metalbinding studies to elucidate the Zn(ll)-binding properties of CP. We report unique optical absorption and EPR spectroscopic signatures for the interfacial His 3Asp and His 4 sites of human CP by using Co(II) as a spectroscopic probe. Zinc competition titrations employing colorimetric and fluorimetric Zn(II) sensors establish that CP coordinates two Zn(II) ions / CP heterodimer. The Ca(ll)-insensitive Zn(ll) sensor ZP4 is used to determine the Kd of CP for Zn(II) in Ca(Il)-deplete and Ca(Il)-replete conditions. These competition titrations afford apparent Kdsitel = 133 58 pM and Kdsite2 = 185 219 nM in the absence of Ca(II). In the presence of excess Ca(Il) these values decrease to Kd,sitel 5 10 pM and Kd,site2 : 240 pM. In vitro antibacterial assays indicate that the metal-binding sites and Ca(ll)-replete conditions are required to inhibit the growth of Gram-negative and Gram-positive bacteria. We propose a model in which Ca(II) ion gradients modulate the antibacterial activity and Zn(Il)-binding properties of human CP. Chapter 3: High-Affinity Manganese Coordination by Human Calprotectin Is Calcium- Dependent and Requires the Histidine-Rich Site at the Dimer Interface. In this chapter, we report that the His 4 motif at the S10OA8/S100A9 dimer interface of CP is required for high-affinity Mn(II) coordination. We identify a low-temperature EPR spectroscopic signal for this site that is consistent with high-spin Mn(II) in an octahedral coordination sphere. This site could be simulated with zero-field splitting parameters D = 270 MHz and EID = 0.30 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that (A8)Hisl7, (A8)His27, (A9)His9l, (A9)His95 and two as-yet unidentified ligands coordinate Mn(ll) at site 2. These studies support a model in which CP responds to Ca(ll) ion gradients to become a potent metal-ion chelator in the extracellular space. Chapter 4: Contributions of the C-terminal Tail of S100A9 to High-Affinity Manganese Binding by Human Calprotectin. This chapter examines the role of the S100A9 C-terminal tail to high-affinity Mn(ll) coordination by human CP. We present a 16-member mutant family with mutations in the S100A9 C-terminal tail (residues 96-114), which houses three histidine and four acidic residues, to evaluate its contribution to Mn(ll) sequestration. These studies confirm that two His residues at positions 103 and 105 complete the octahedral coordination sphere of CP in solution. Appendix 1: Sequence Alignments of Transition-Metal Binding S100 Proteins. Sequence alignments of S100A7, S100A8, S100A9, S100A12, S100A15, and S100B proteins from multiple organisms are presented. Appendix 2: Characterization of CP Mutant Proteins by Circular Dichroism and Analytical Size Exclusion Chromatography. Additional characterization of CP and mutant proteins employed in Chapters 2-4 is presented. Appendix 3: Structures of Sensors Used In this Work. The structures of Zincon, MagFura-2, Zinpyr-1, and Zinpyr-4 are presented. Appendix 4: Manganese Binding Properties of Human Calprotectin under Conditions of High and Low Calcium. This appendix represents a collaborative work with the Drennan Lab (MIT) and Britt Lab (UC Davis) to study the Mn(Il)-CP complex in low- and high-Ca(II) conditions. We report a crystal structure of Mn(Il)-, Ca(Il)-, and Na(l)-bound CP with Mn(II) exclusively coordinated to the His6 motif. Electron spin-echo envelope modulation and electron-nuclear double resonance experiments demonstrate that the six coordinating histidine residues are spectroscopically equivalent. The observed 15N ( = %/h)y perfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating E-nitrogen of the imidazole ring of each histidine (A = [3.45, 3.71, 5.91] MHz) and the distal 6-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the affinity of CP for Mn(II) drops by two to three orders of magnitude, and Mn(II) coordinates to the His6 site as well as other sites on the protein.by Megan Brunjes Brophy.Ph. D. in Biological Chemistr
Manganese and Microbial Pathogenesis: Sequestration by the Mammalian Immune System and Utilization by Microorganisms
ABSTRACT: Bacterial and fungal pathogens cause a variety of infectious diseases and constitute a significant threat to public health. The human innate immune system represents the first line of defense against pathogenic microbes and employs a range of chemical artillery to combat these invaders. One important mechanism of innate immunity is the sequestration of metal ions that are essential nutrients. Manganese is one nutrient that is required for many pathogens to establish an infective lifestyle. This review summarizes recent advances in the role of manganese in the hostāpathogen interaction and highlights Mn(II) sequestration by neutrophil calprotectin as well as how bacterial acquisition and utilization of manganese enables pathogenesis. Metals ions are essential for the proliferation of allkingdoms of life and perform a variety of structural and chemical tasks. In order to establish a virulent lifestyle, pathogens must acquire and concentrate metal ions from the host.1ā3 The mammalian innate immune system has evolved t
Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin
Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His3Asp and His4 sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His[subscript 3]Asp and His[subscript 4] sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K[subscript d1] = 133 Ā± 58 pM and K[subscript d2] = 185 Ā± 219 nM for CP in the absence of Ca(II). These values decrease to K[subscript d1] ā¤ 10 pM and K[subscript d2] ā¤ 240 pM in the presence of excess Ca(II). The K[subscript d1] and K[subscript d2] values are assigned to the His[subscript 3]Asp and His[subscript 4] sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.National Institutes of Health (U.S.) (Office of the Director, NIH Grant DP2OD007045)Searle Scholars ProgramMassachusetts Institute of Technology. Center for Environmental Health Sciences (NIH Grant P30-ES002109)Massachusetts Institute of Technology. Dept. of Chemistr
High-Affinity Manganese Coordination by Human Calprotectin Is Calcium-Dependent and Requires the Histidine-Rich Site Formed at the Dimer Interface
Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of CP. We demonstrate that the unusual Hisā motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters D = 270 MHz and E/D = 0.30 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The HisāAsp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the Hisā site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the Hisā site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth
Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin
Calprotectin (CP) is an antimicrobial protein produced
and released
by neutrophils that inhibits the growth of pathogenic microorganisms
by sequestering essential metal nutrients in the extracellular space.
In this work, spectroscopic and thermodynamic metal-binding studies
are presented to delineate the zinc-binding properties of CP. Unique
optical absorption and EPR spectroscopic signatures for the interfacial
His<sub>3</sub>Asp and His<sub>4</sub> sites of human calprotectin
are identified by using CoĀ(II) as a spectroscopic probe. Zinc competition
titrations employing chromophoric ZnĀ(II) indicators provide a 2:1
ZnĀ(II):CP stoichiometry, confirm that the His<sub>3</sub>Asp and His<sub>4</sub> sites of CP coordinate ZnĀ(II), and reveal that the ZnĀ(II)
affinity of both sites is calcium-dependent. The calcium-insensitive
ZnĀ(II) competitor ZP4 affords dissociation constants of <i>K</i><sub>d1</sub> = 133 Ā± 58 pM and <i>K</i><sub>d2</sub> = 185 Ā± 219 nM for CP in the absence of CaĀ(II). These values
decrease to <i>K</i><sub>d1</sub> ā¤ 10 pM and <i>K</i><sub>d2</sub> ā¤ 240 pM in the presence of excess
CaĀ(II). The <i>K</i><sub>d1</sub> and <i>K</i><sub>d2</sub> values are assigned to the His<sub>3</sub>Asp and His<sub>4</sub> sites, respectively. <i>In vitro</i> antibacterial
activity assays indicate that the metal-binding sites and CaĀ(II)-replete
conditions are required for CP to inhibit the growth of both Gram-negative
and -positive bacteria. Taken together, these data provide a working
model whereby calprotectin responds to physiological CaĀ(II) gradients
to become a potent ZnĀ(II) chelator in the extracellular space
Biochemical and Spectroscopic Observation of Mn(II) Sequestration from Bacterial Mn(II) Transport Machinery by Calprotectin
Human calprotectin (CP, S100A8/S100A9 oligomer) is a metal-sequestering host-defense protein that prevents bacterial acquisition of Mn(II). In this work, we investigate Mn(II) competition between CP and two solute-binding proteins that Staphylococcus aureus and Streptococcus pneumoniae, Gram-positive bacterial pathogens of significant clinical concern, use to obtain Mn(II) when infecting a host. Biochemical and electron paramagnetic resonance (EPR) spectroscopic analyses demonstrate that CP outcompetes staphylococcal MntC and streptococcal PsaA for Mn(II). This behavior requires the presence of excess Ca(II) ions, which enhance the Mn(II) affinity of CP. This report presents new spectroscopic evaluation of two Mn(II) proteins important for bacterial pathogenesis, direct observation of Mn(II) sequestration from bacterial Mn(II) acquisition proteins by CP, and molecular insight into the extracellular battle for metal nutrients that occurs during infection.National Institutes of Health (U.S.) (Grant R01GM118695)National Institutes of Health (U.S.) (Grant R01GM104543)National Science Foundation (U.S.) ( Grant 0070319)National Institutes of Health (U.S.) (Grant P30-ES002109
Contributions of the S100A9 C-Terminal Tail to High-Affinity Mn(II) Chelation by the Host-Defense Protein Human Calprotectin
Human calprotectin (CP) is an antimicrobial protein that coordinates Mn(II) with high affinity in a Ca(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96ā114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn(II) coordination sphere in solution, affording an unprecedented biological His6 site. These solution studies are in agreement with a Mn(II)-CP crystal structure reported recently (Damo, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3841). Remarkably high-affinity Mn(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103ā105 to 104ā106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against Escherichia coli and Staphylococcus aureus. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn(II) binding; the S100A7, S100A9(C3S), S100A12, and S100B homodimers do not exhibit such Mn(II)-binding capacity.Searle Scholars ProgramNational Institutes of Health (U.S.) (Massachusetts Institute of Technology. Center for Environmental Health Sciences. NIH P30-ES002109)Stephen J. Lippard Fellowship FundEĢcole normale supeĢrieure (Cachan, France
Biochemical and Functional Evaluation of the Intramolecular Disulfide Bonds in the Zinc-Chelating Antimicrobial Protein Human S100A7 (Psoriasin)
Human S100A7 (psoriasin) is a metal-chelating protein expressed by epithelial cells. It is a 22-kDa homodimer with two EF-hand domains per subunit and two transition-metal-binding His[subscript 3]Asp sites at the dimer interface. Each subunit contains two cysteine residues that can exist as free thiols (S100A7[subscript red]) or as an intramolecular disulfide bond (S100A7[subscript ox]). Herein, we examine the disulfide bond redox behavior, the Zn(II) binding properties, and the antibacterial activity of S100A7, as well as the effect of Ca(II) ions on these properties. In agreement with prior work [Hein, K. Z., et al. (2013) Proc. Natl. Acad. Sci. U. S. A. 112, 13039-13044], we show that apo S100A7ox is a substrate for the mammalian thioredoxin system; however, negligible reduction of the disulfide bond is observed for Ca(II)- and Zn(II)-bound S100A7[subscript ox]. Furthermore, metal binding depresses the midpoint potential of the disulfide bond. S100A7[subscript ox] and S100A7[subscript red] each coordinate 2 equiv of Zn(II) with subnanomolar affinity in the absence and presence of Ca(II) ions, and the cysteine thiolates in S100A7[subscript red] do not form a third high-affinity Zn(II) site. These results refute a prior model implicating the Cys thiolates of S100A7[subscript red] in high-affinity Zn(II) binding [Hein, K. Z., et al. (2013) Proc. Natl. Acad. Sci. U. S. A. 112, 13039-13044]. S100A7[subscript ox] and the disulfide-null variants show comparable Zn(II)-depletion profiles; however, only S100A7[subscript ox] exhibits antibacterial activity against select bacterial species. Metal substitution experiments suggest that the disulfide bonds in S100A7 may enhance metal sequestration by the His[subscript 3]Asp sites and thereby confer growth inhibitory properties to S100A7[subscript ox]. Keywords: disulfides; redox reactions; peptides and proteins; monomersIonsNational Science Foundation (U.S.) (CHE-1352132
Contributions of the S100A9 CāTerminal Tail to High-Affinity Mn(II) Chelation by the Host-Defense Protein Human Calprotectin
Human
calprotectin (CP) is an antimicrobial protein that coordinates
MnĀ(II) with high affinity in a CaĀ(II)-dependent manner at an unusual
histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface.
We present a 16-member CP mutant family where mutations in the S100A9
C-terminal tail (residues 96ā114) are employed to evaluate
the contributions of this region, which houses three histidines and
four acidic residues, to MnĀ(II) coordination at site 2. The results
from analytical size-exclusion chromatography, MnĀ(II) competition
titrations, and electron paramagnetic resonance spectroscopy establish
that the C-terminal tail is essential for high-affinity MnĀ(II) coordination
by CP in solution. The studies indicate that His103 and His105 (HXH
motif) of the tail complete the MnĀ(II) coordination sphere in solution,
affording an unprecedented biological His<sub>6</sub> site. These
solution studies are in agreement with a MnĀ(II)-CP crystal structure
reported recently (Damo, S. M.; et al. <i>Proc. Natl. Acad. Sci.
U.S.A. </i> <b>2013</b>, <i>110</i>, 3841). Remarkably
high-affinity MnĀ(II) binding is retained when either H103 or H105
are mutated to Ala, when the HXH motif is shifted from positions 103ā105
to 104ā106, and when the human tail is substituted by the C-terminal
tail of murine S100A9. Nevertheless, antibacterial activity assays
employing human CP mutants reveal that the native disposition of His
residues is important for conferring growth inhibition against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>. Within the S100 family, the S100A8/S100A9 heterooligomer is essential
for providing high-affinity MnĀ(II) binding; the S100A7, S100A9Ā(C3S),
S100A12, and S100B homodimers do not exhibit such MnĀ(II)-binding capacity