by: Deepika Satish

Abstract

Studying the evolution of globins and observing patterns of oxygen migration is one method of determining how nature has selectively chosen properties for oxygen storage and transport. In this project, we have studied Scapharca inaequivalvis Hemoglobin I (ScHbI), a dimeric protein found in Scapharca inaequivalvis clams, to map its internal migration of oxygen and compare the observed pathway to the distal histidine ‘gate’ pathway found in both monomeric mammalian myoglobins and tetrameric adult human hemoglobin. For ScHbI, this putative distal histidine gate (His-69(E7)) appears to be closed by the hemoglobin’s inverted quaternary structure; thus, we tested if it was the ligand migration pathway in ScHbI by first changing the size and polarity at and around the distal histidine gate and then examining the effects on oxygen association, dissociation, and affinity. Our results strongly suggest that, despite facing the dimer interface, ScHbI’s E7 residue functions as the gate for diatomic ligand capture in the distal pocket, thus having a similar oxygen migration pathway as mammalian myoglobins and human hemoglobins. By studying ScHbI and other model hemoglobins, we are building up a set of rules that govern the function of all hemoglobins. These rules can then be employed to optimize the properties of recombinant human hemoglobin and to engineer a hemoglobin-based oxygen carrier or blood substitute for the treatment of hemorrhagic shock due to severe blood loss.

Blood Substitutes

As the demand for whole donated blood increases every day, the need for blood substitutes becomes more and more important for the treatment of hemorrhagic shock under emergency situations. Over the years, much effort has been expended on making safe, effective blood substitutes, but none have been able to perfectly emulate human hemoglobin (HbA) within intact red blood cells. A hemoglobinbased oxygen carrier (HBOC) named PolyHeme® is one of the few oxygen-carrying blood substitutes to complete a phase III trial. However, it failed to receive FDA approval in April 2009 because the studies revealed that “Polyheme® places the patients at a higher risk of significant adverse events”,1 such as heart attacks and myocardial infarctions.2 Another hemoglobin-based blood substitute known as Hemopure ® is the first and only oxygen-carrying blood substitute to be approved for human use in South Africa. However, in the United States, analyses of the phase III human trials conducted using Hemopure® were brought into question, and in mid-2002, the FDA mandated that further animal trials on nitric oxide scavenging and blood pressure effects be conducted before any new human clinical trials could resume.3,4 This brings to view just one of the many obstacles faced when developing blood substitutes. When hemoglobin- based blood substitutes have low oxygen association, they tend to bind nitric oxide (known as NO scavenging), which triggers vasoconstriction. This results in hypertension, a major adverse effect of current hemoglobin-based blood substitutes.

Dr. John S. Olson’s laboratory has focused on engineering recombinant hemoglobin (Hb) that has fewer blood pressure side effects and increased oxygen delivery efficiency. His laboratory has conducted multiple molecular biophysics studies exploring the optimization of the properties required for a successful blood substitute. Furthermore, recombinant hemoglobins from multiple organisms are being studied to understand how their unique structures affect their oxygen binding properties and to apply this knowledge in developing strategies for engineering extracellular hemoglobin as a blood substitute.

Engineering Recombinant Hemoglobin

Designing recombinant hemoglobin for use as a blood substitute necessitates the creation of a mutant with moderately low oxygen affinity, large oxygen exchange rates, high subunit cooperativity, and minimum rates of nitric oxide scavenging.5 In order to synthetically optimize the function of recombinant hemoglobin in attaining these properties, we require a general understanding of how globins evolved in nature under selective pressure for cooperative and rapid oxygen binding and release. A general knowledge base will enable us to develop a set of rules governing the speed of ligand (oxygen and carbon monoxide) migration into the protein and the stereochemistry of iron-ligand bond formation within the globin active site. With a progressively better understanding of oxygen migration paths within these proteins, we will be able to understand how gases migrate within proteins in general and to identify patterns of pathways for different families of hemoglobins.6 Most importantly, we will be able to predict oxygen pathways in various types of hemoglobin and modify them by protein engineering. Although many different types of oxygen pathways in globins have been mapped thus far, the best understood is sperm whale myoglobin (SwMb). Its oxygen interactions have been studied extensively, and it has served as the model system of protein dynamics for many years. 7,8 While SwMb studies have helped shed light on the basics of oxygen migration, a key feature of the globins that has not been extensively explored is if or how cooperative interactions between subunits alter ligand pathways. Multimers, such as HbA, often display cooperativity through inter-monomer conformational changes. 9,10 In this study, hemoglobin from the mollusk Scapharca inaequivalvis (ScHbI) was chosen as the model system because it represents the simplest allosteric system of two identical subunits in which one subunit alters the other’s ligand affinity at a chemically identical active site and because the interface appears to block the distal histidine gate found in mammalian Mbs and Hbs. 11

Scapharca inaequivalvis Hb I

Mammalian hemoglobin is a complex tetrameric protein that delivers oxygen to and transports carbon dioxide away from various tissues in the body (Figure 1A). The protein has four subunits, each containing a prosthetic group known as heme. Each heme molecule contains one iron that binds to a single diatomic oxygen molecule. In SwMb and HbA, ligands gain access to this heme located in the ligand binding pocket by moving through a channel created by upward and outward movement of the distal histidine E7 side chain. This mechanism for ligand entry is known as the His(E7) gate. ScHbI is thought to have a His(E7) gate similar to that in Mb and HbA.8,12 However, the His(E7) gates of this dimeric hemoglobin appear to be closed by its inverted quaternary structure, as each subunit seems to cover and block the outward movement its adjacent subunit’s His(E7) gate. Thus, to bind to the heme within the distal pocket, oxygen would have to first migrate into the dimer interface, and then proceed past the His(E7) gate (Figure 1B),13 unlike in HbA, which has ‘open’ (E7) gates that are not blocked by the molecule’s own subunits (Figure 1A). Thus, whether oxygen still enters and exits ScHbI through the E7 gate is an open question.7

In this project, mutagenesis has been conducted at five key helical positions (E7, CD3, CD4, F4, and B3), which are either at the E7 gate or within the ligand pocket, to create the library of ScHbI mutants, most of which are shown in Figure 3. These mutations were intended to demonstrate a wide range of effects, all of which were chosen to determine if the E7 gate has a role in ligand migration. If oxygen enters through the E7 gate, then varying the size and polarity of the amino acids at the putative gate (E7) should show large effects on the rate of ligand association; altering the ease of opening of the gate and the amount of internal water that must be displaced before oxygen can bind would result in measureable changes in oxygen association and dissociation.14 Altering amino acids within the distal pocket (at positions CD3 and CD4) should affect the ease of movement of the His(E7) gate and thus the rate of migration of ligands into and out of the distal pocket. Lastly, mutations to reduce cooperativity (at position F4) and to render a monomeric form of the protein (at position B3) should simplify the model such that the role of the E7 gate can be further elucidated.15,16,17 The recombinant hemoglobin protein was constructed based on the protocol prescribed by Summerford et al.18 By observing the effects of these changes in the oxygen binding rate and equilibrium constants of the hemoglobin and identifying the trends that depend on the size and flexibility of the side chain at the E7 position, we can determine whether the distal histidine gate is the route of ligand migration in ScHbI.

Mapping Ligand Migration

Time courses for O2 and CO binding were measured and used to determine ligand association (k’) and dissociation (k) rates and equilibrium association constants (K). By looking at trends in the oxygen rate and affinity constants for the library of ScHbI mutants in terms of the size and polarity of the altered side chains, we can map the pathway of oxygen migration and examine the strength of electrostatic and steric interactions with the bound ligand.

In these kinetic experiments, CO is photodissociated from the heme group of ScHbI in the presence of O2 by a flash tube-driven dye laser, which provides an intense light pulse for ~0.5 μs at 577 nm. When the absorbance changes in the sample are followed at 436 nm, we can observe the formation of deoxyHb (hemoglobin without any bound ligand) during the laser excitation pulse, and then follow its decay as ligands rebind from solvent in a bimolecular process. The initial fast bimolecular rebinding phase (kf) represents rebinding of O2 and some CO at the rate given in the equations below. If the absorbance changes are followed at 425 nm, a slow unimolecular phase (ks) is observed and represents the displacement of oxygen that was bound in the fast phase by carbon monoxide, which has a greater affinity for the protein. Expressions for the rate of this slow phase and the overall oxygen affinity constant, which is calculated from the association (k’O2) and dissociation (kO2) constants, are also given below:

Sets of k’O2, kO2, and KO2 were obtained for the different mutants, after which trends and correlations with increasing size or polarity were examined.

Analysis of the kinetic data for ScHbI suggests that the major pathway for ligand entry and exit in ScHbI is through the E7 gate despite the gate’s apparent closure by the adjacent subunit. In general, increasing the size of the amino acid at the E7 position decreases the rate of oxygen association up to 10-fold, indicating the involvement of the E7 gate in oxygen migration. This is further supported by the fact that changing the polarity of the E7 amino acid has a great effect on the rates of both ligand association and dissociation. Polar amino acids that are capable of donating a hydrogen bond markedly stabilize bound oxygen. However, these same amino acids stabilize water in the binding pocket of deoxyScHbI and slow the rate of ligand association. Thus, when histidine, asparagine, and glutamine are present at the E7 position, there is a complex tradeoff between the effects of size and polarity of the amino acid on oxygen association and dissociation rates. Mutations at the CD4, CD3, F4, and B3 positions also indicate that the main path of ligand migration is through the dimer interface and the E7 gate.

In general, the pattern of mutagenesis effects observed for ScHbI resembles those reported previously for the subunits of HbA and SwMb, which suggests that the distal histidine moves outward to allow diatomic ligand entry and exit.12,8. Thus, our work with ScHbI indicates that the basic rules governing the ligand migration and affinity in ScHbI are identical to those for mammalian Hbs and Mbs and apply in general to all globins with a distal histidine.

Future Work

While the experiments done so far on ScHbI all indicate that the E7 gate is the route of ligand migration, a few more mutants need to be made to fully demonstrate the range of effects that the mutations have on the rate of oxygen association and dissociation. This project has enabled us to further understand the evolution of globins and helped us to elucidate a model on which future experiments can be based, but much work still needs to be done to completely understand animal hemoglobins and establish principles for globin design. Properties required for a hemoglobin-based blood substitute include moderately low oxygen affinity, high oxygen exchange rates, high subunit cooperativity, discrimination against CO binding, low rates of nitrogen oxide reaction, resistance to autooxidation, resistance to reactions of H2O2, resistance to denaturation, and high affinity to heme,5 of which only the first three properties were studied following the methods used in this project. However, all of these key ideas must undergo more extensive investigation, especially in HbA. Continuing research of hemoglobins of other organisms is also crucial to understanding the patterns for all of these properties throughout evolution.

Deepika Satish has worked in Dr. John S. Olson’s lab where she has constructed recombinant ScHbI, performed kinetic tests on the recombinant protein, and analyzed the trends of the kinetic tests in relation to the HbA. Previous such hemoglobin research conducted in the Olson lab has yielded ideas that have led to the construction of safer second-generation hemoglobin-based blood substitutes by the Baxter Hemoglobin Therapeutics.5 Continued research in this area will hopefully contribute more elucidations and ideas for more efficient and effective blood substitute construction. Deepika is a sophomore Biochemistry and Cell Biology major at Will Rice College ’12.

by: Navonil Ghosh

Abstract

Angiogenesis, the growth of new blood vessels from preexisting vasculature, is a significant physiological process in living organisms. Vascular endothelial growth factors, or VEGF, and associated receptors are key effectors during the formation of blood vessels. Because any defects or abnormalities in vessel formation can result in a variety of vascular diseases and disorders, an intimate understanding of the developmental pathways that regulate angiogenesis could elucidate the causes of these diseases and reveal possible treatments or preventative measures. Thus, scientists are exploring a number of angiogenic signaling pathways and effectors of these pathways, particularly VEGF. By manipulating these pathways and analyzing the resulting phenotypic effects on blood vessel formation, researchers can determine the effectiveness of angiogenic regulatory molecules as therapeutic agents for vascular disorders.

Introduction

The vasculature of vertebrate organisms is regulated by a number of signaling pathways that affect vascular endothelial cells, which comprise the inner lining of blood vessels. These pathways govern both the growth and patterning of blood vessel formation. Any disruption to the developmental cues necessary for proper growth can result in defective vascular formation that can lead to a number of diseases including psoriasis, rheumatoid arthritis, and cancer. Because the blood vessels of the vascular system are intricately linked to the survival of all vertebrates, a vast number of clinical and developmental studies have focused on angiogenesis.

Although the term was first used by Dr. John Hunter in 1787 to describe blood vessel growth in reindeer antlers, angiogenesis entails the formation of new capillary blood vessels in all vertebrates and is a major area of developmental study.1 In healthy vertebrate organisms, angiogenesis is a key component to healing wounds and restoring circulation to damaged tissue. The process is controlled in a healthy organism by a series of angiogenesis-stimulating growth factors and angiogenesis inhibitors, which serve as the “on” and “off” switches for blood vessel growth.2 The angiogenic process was thus proposed to have a therapeutic role by means of new vessel induction in damaged tissue or inhibition of vascular formation in pathological pathways involving abnormal vessel growth. According to the Angiogenesis Foundation, approximately 500 million patients in Western nations alone could benefit from either proangiogenic or antiangiogenic therapy. As a result, over $4 billion in medical research has been devoted to developing angiogenic-based therapeutic drugs.1

Angiogenic Vessel Formation

Angiogenesis is dependent on a signaling cascade to induce vascular development (Fig. 1).1 The first step of the angiogenic process is the release of angiogenic growth factors that bind to the membrane-bound receptors of nearby vascular endothelial cells. These activated endothelial cells then start producing and secreting enzymes that degrade the basement membrane, a sheath-like covering of blood vessels. They also initiate an intracellular signaling cascade that prompts the formation of other building blocks of endothelial cell formation. The endothelial cells divide and pass through the dissolved openings of the basement membrane to where the new vessel will form. The sprouting of the newly developing vessel is aided by adhesion molecules known as integrins that bind and help pull the vessel into place. Meanwhile, other enzymes such as matrix metalloproteinases degrade the surrounding extracellular matrix to make room for the growing vessel. The endothelial cells then join together to form individual capillary sprouts, which are stabilized by smooth muscle cells and pericyte muscle cells. Blood circulation begins when the individual tubes join together to form blood vessel loops.

Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is one of the most critical effectors involved in angiogenesis. VEGF, a family of tyrosine kinases, stimulates a variety of signaling pathways that regulate the growth of endothelial cells.3 The VEGF system involves a series of ligands and receptors. The secreted ligands include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF and the receptors include VEGF-R1, VEGF-R2, VEGF-R3. Through autophosphorylation by the tyrosine kinases of the VEGF family, the VEGF receptors activate cellular signaling pathways that ultimately control migration, survival, and proliferation of vascular endothelial cells. Because VEGF plays such an intimate role in angiogenesis, it is highly utilized in a variety of scientific studies involving other angiogenic factors.4 A recent study, for example, shows that angiomodulin, a modulator of VEGF-dependent vascular formation, has increased expression in human tumor tissue.5 Although further examination of angiomodulin’s role in cancer progression is needed, this could mark a step in evaluating angiogenic pathways in a therapeutic role for cancer.

The Zebrafish Model

There are a variety of in vivo models available for angiogenesis (Table 1).6 The zebrafish model organism, Danio rerio, is particularly well-suited for the analysis of developing vasculature structures for a variety of reasons. It produces a large clutch size of transparent, external embryos that allow easy, accessible observation from an early age.7 Furthermore, the rapid development of zebrafish embryos also allows for timely analysis and easy reproduction of experimental results. The genetic pathways of zebrafish share many angiogenic mechanisms and genes with other vertebrates, and because these pathways can be easily manipulated, the zebrafish can serve as a useful tool for analyzing the development of vasculature in vertebrate organisms in general.8 Moreover, the vasculature itself can be easily observed through the use of transgenics, allowing the intersegmental, subintestinal, and cranial vessels of the organism to be easily examined (Fig 2.).9 By virtue of these advantages, the zebrafish is a highly used model organism by scientists studying the processes that regulate blood vessel formation.

In order to reproduce defects in the vasculature of the zebrafish model organism, scientists rely on a number of effective assays and experimental procedures. In order to visibly examine the blood vasculature of zebrafish, transgenic zebrafish with fluorescent vessels have been developed. Fluorescent expression through the insertion of the green fluorescent protein (GFP) construct into fli-eGFP, mTie2-GFP, and flk-GFP promoters, which are expressed in vascular endothelial cells, allows the entire vasculature of the organism to be visible.8 Microinjection of mRNA and morpholinos can be used to examine a target gene’s role in vascular development by showing the effects of altering normal gene expression.7 Injecting mRNA overexpresses the target gene, while the morpholinos block translation to knockdown the gene. Injections into transgenic zebrafish allow the effects of overexpression and knockdown to be examined in great detail. Further genetic analysis can be done through in situ hybridizations, which uses antibodies to stain a complementary strand of RNA that binds to a target gene. This allows for the identification of changes in the expression of other genes through alteration of the original target gene.

Angiogenic-Dependent Diseases

Angiogenesis-based pathological conditions can occur when the body loses control over the angiogenic process and blood vessel formation progresses either excessively or insufficiently .1 In diseases dependent on excessive angiogenesis, diseased cells release excessive amounts of angiogenic growth factors that cause abnormal proliferation of the endothelial cells that make up newly developing vessels. These excessively sprouting blood vessels damage healthy tissue while continuing to nourish the diseased tissue. Conditions related to an overgrowth of blood vessels include macular degeneration, diabetic blindness, and psoriasis. Abnormal vessels can even allow cancerous growths to metastasize by providing the means for tumor cells to reach healthy organs. In the case of insufficient angiogenic vessel growth, the body cannot produce enough angiogenic growth factors for proper vessel formation. The lack of adequate blood vessel growth affects tissue perfusion and can cause necrosis. Insufficient angiogenesis is involved with both coronary artery disease and stroke.

Angiogenic Therapy and Inhibition

Because so many pathological conditions have a tie to abnormal blood vessel formation, scientists have proposed that angiogenesis may have therapeutic purposes. Therapeutic angiogenesis generally involves turning the genes controlling the process, or using angiogenesis – stimulating growth factors to allow for the growth of new blood vessels.1 Angiogenic stimulation targets vascular endothelial cells by using cytokine growth factors, such as those of the VEGF family. The resulting increase in endothelial cell proliferation enhances vessel formation and blood profusion to damaged, or ischemic, tissue. This proangiogenic stimulation is induced in order to enhance blood flow to injured tissue, transport survival factors to areas in need of tissue repair, activate populations of regenerative stem cells, and ultimately restore tissue to its normal state.

A number of clinical drugs and interventions have been developed to treat pathologies through therapeutic angiogenesis. The three diseases most commonly treated using angiogenic therapy are chronic wounds, such as arterial or venous ulcers, peripheral arterial disease, and ischemic heart disease.1 To treat these diseases, angiogenesis can be stimulated through therapeutic angiogenic drugs. An FDA-approved growth-factor based drug known as rhPDGF is a recombinant protein medication used to treat diabetic ulcers. Systems such as autologel or SmartPReP allow for the treatment of injured tissue using autologous blood samples, which contain the natural growth factors and cytokines needed to stimulate healing through angiogensis. 10, 11 Early clinical trials of other angiogenic stimulators have shown evidence of increased blood flow and function of the left ventricle. This is a promising therapeutic tool for treating coronary heart disease. Angiogenesis can also be promoted without relying on drugs or medication. Negative pressure wound therapy stimulates blood vessel growth through micro-deformations of tissue that respond with signal transduction leading to epithelial proliferation. Other methods include the MIST ultrasound, a low-frequency and low-intensity procedure that stimulates endothelial cells and enhances blood perfusion, and Hyperbaric Oxygen (HBO), which increases expression of VEGF and endothelial progenitor cells.12, 1

Inhibition of angiogenesis can also have therapeutic properties, for it can prevent feeding of diseased tissue such as cancerous tumors. Anti-angiogenic agents inhibit cell signaling pathways necessary for vessel growth. These inhibitors include three major varieties: monoclonal antibodies, tyrosine kinase inhibitors, and inhibitors of mammalian targets of rapamycin (mTOR).1 Monoclonal antibodies bind to VEGF to prevent it from binding in turn to VEGF receptors, thus interrupting the proliferation of endothelial cells. Small molecule tyrosine kinase inhibitors interrupt pathways involving a number of tyrosine kinase growth factor receptors, including VEGF receptors. mTOR inhibitors intercept the PI3/AKT pathway necessary for tumor proliferation. 13 All three methods act by inhibiting a pathway necessary for endothelial proliferation and thus prevent angiogenic nourishment of diseased or cancerous tissue.

Conclusions

Therapeutic proangiogenic and antiangiogenic applications have shown potential in alleviating a number of vascular-based pathologies. The zebrafish developmental model remains a valuable tool for analyzing vascular formation and potential angiogenic therapies. As our understanding of angiogenesis increases, advancements in therapeutic uses for the process will have an important impact on clinical treatment of vascular pathologies.

Acknowledgements

I would like to thank Dr. Daniel Wagner for introducing me to the angiogenic and zebrafish research. I would also like to thank Jane Li, Casey O’Grady, Eric Kim, and Dr. Amina Qutub for their feedback and advice upon editing my article.

References

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Navonil Ghosh is a sophomore from Hanszen College majoring in Biochemistry and Cell Biology. Under the supervision of Dr. Daniel Wagner of the Biochemistry and Cell Biology Department at Rice University, Ghosh is investigating the role of the aggf1 gene in angiogenesis. Angiogenic Factor with G patch and FHA domains 1 (AGGF1) has been associated with the VEGF pathway, as well as a vascular birth defect known as Klippel-Trenaunay-Weber Syndrome. Thus, aggf1 is hypothesized as an important regulator of vascularization in zebrafish. Ghosh is analyzing the effects of overexpression and knockdown of aggf1 on the vasculature of the zebrafish model in order to better understand the role of aggf1 in angiogenesis and evaluate its therapeutic potential.