Benoit J. Smagghe

NO Dioxygenase Activity in Hemoglobins Is Ubiquitous In Vitro, but Limited by Reduction In Vivo

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to “hexacoordinate” hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O2 binding.

Review: Correlations between oxygen affinity and sequence classifications of plant hemoglobins

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full‐length globins with the classical eight helix Mb‐like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O2 binding (∼250 μM−1 s−1), moderately high rates of O2 dissociation (∼5–15 s−1), and high oxygen affinity (Kd or P50 ≈ 50 nM). These properties both facilitate O2 diffusion to respiring N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (KHisE7 ≈ 2), moderate rates of O2 binding (∼25 μM−1 s−1), very small rates of O2 dissociation (∼0.16 s−1), and remarkably high O2 affinities (P50 ≈ 2 nM), suggesting a function involving O2 and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (KHisE7 ≈ 100), low rates of O2 binding (∼1 μM−1 s−1), moderately low O2 dissociation rate constants (∼1 s−1), and moderate, Mb‐like O2 affinities (P50 ≈ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2‐vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117–heme 2‐vinyl bond because it also displays endogenous bis‐histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117–heme 2‐vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117–heme 2‐vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117–heme 2‐vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.

Immunolocalization of non-symbiotic hemoglobins during somatic embryogenesis in chicory.

Hemoglobins are ancient O2-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.

MUC1* Ligand, NM23-H1, Is a Novel Growth Factor That Maintains Human Stem Cells in a More Naïve State

We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more “naïve” state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cells inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cells exit from pluripotency.

Glutathione-S-Transferase is Detected During Somatic Embryogenesis in Chicory.

Glutathione S-tranferases (GSTs) are a heterogeneous family of proteins, which perform diverse pivotal catalytic and non-enzymatic functions during plant development and in plant stress responses. Previous studies have shown that a GST activity (EC 2.5.1.18) is closely linked with the precocious phases of somatic embryogenesis in leaf tissues of an interspecific chicory hybrid (Cichorium intybus L. var. sativa x C. endivia L.var. latifolia). In order to learn more about the involvement of this enzyme in this process, in situ-hybridization as well as immunolocalization were performed in parallel. GST-mRNAs and proteins were co-localized in small veins, particularly in young protoxylem cell walls. During cell reactivation, the in situ and protein signals became less intense and were associated with chloroplasts. The GST-mRNAs and corresponding proteins were not always co-localized in the same tissues. While high amounts of transcripts could be detected in multicellular embryos, the proteins were not well labeled. Our results indicated that GSTs belong to a complex anti-oxidant mechanism within the cell, and also at the cell wall level. GSTs presence in reactivated cell and multicellular embryos is discussed in relation to redox cell status.

A Primitive Growth Factor, NME7AB, Is Sufficient to Induce Stable Naïve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation

Scientists have generated human stem cells that in some respects mimic mouse naïve cells, but their dependence on the addition of several extrinsic agents, and their propensity to develop abnormal karyotype calls into question their resemblance to a naturally occurring “naïve” state in humans. Here, we report that a recombinant, truncated human NME7, referred to as NME7AB here, induces a stable naïve‐like state in human embryonic stem cells and induced pluripotent stem cells without the use of inhibitors, transgenes, leukemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), feeder cells, or their conditioned media. Evidence of a naïve state includes reactivation of the second X chromosome in female source cells, increased expression of naïve markers and decreased expression of primed state markers, ability to be clonally expanded and increased differentiation potential. RNA‐seq analysis shows vast differences between the parent FGF2 grown, primed state cells, and NME7AB converted cells, but similarities to altered gene expression patterns reported by others generating naïve‐like stem cells via the use of biochemical inhibitors. Experiments presented here, in combination with our previous work, suggest a mechanistic model of how human stem cells regulate self‐replication: an early naïve state driven by NME7, which cannot itself limit self‐replication and a later naïve state regulated by NME1, which limits self‐replication when its multimerization state shifts from the active dimer to the inactive hexamer. Stem Cells 2016;34:847–859

Abstract 3330: MUC1* targeting CAR T

Purpose: Over 75% of all solid tumor cancers are characterized by aberrant expression of MUC1. Yet to date, no effective MUC1 targeting therapeutic has succeeded. The purpose of this body of work was to develop an antibody therapeutic that would effectively treat MUC1 positive solid tumor cancers, without damaging healthy MUC1 positive tissues. Experimental Procedures: MUC1 is a heavily glycosylated transmembrane protein whose expression is restricted to the apical border of healthy epithelial tissues but uniformly expressed over cancerous tissues. We discovered that the cancer-associated form of MUC1 is transmembrane cleavage product that remains after cleavage and release of the bulk of the extracellular domain. We named cleaved MUC1, MUC1* (muk 1 star), and showed that it is a growth factor receptor that is activated by ligand-induced dimerization of its small extracellular domain. We also showed that 100% of pluripotent human stem cells express MUC1* but cleavage stops with the onset of differentiation. By studying stem cells and cancer cells in parallel, we sought to develop antibodies that would only recognize MUC1* as it is expressed on cancerous tissues but would not recognize MUC1* as it exists on some healthy cells and tissues. Summary New Data: We previously showed that we had developed antibodies that recognized the cleaved, growth factor receptor form, MUC1* but not full length MUC1. However, MUC1 is cleaved to a healthy form of MUC1* on, for example, pluripotent stem cells, hematopoietic stem cells and stem cells of intestinal crypts. We now report that we have developed monoclonal antibodies that only recognize the cancerous form of MUC1* and do not recognize these healthy forms of MUC1*. To demonstrate that we have succeeded in deciphering the differences between cancerous MUC1* and healthy MUC1*, we also developed a set of monoclonal antibodies that only recognize healthy MUC1*. Thousands of human cancerous versus normal tissue specimens attest to the specificity of these antibodies. Cancer-specific anti-MUC1* antibodies (Fabs) alone blocked the growth of MUC1* positive breast and hormone refractory prostate cancers in animals, with no detectable adverse effects. On the basis of tissue safety studies and efficacy studies, a clinical candidate antibody MNC2 has been identified, humanized and incorporated into more than 20 CAR constructs. huMNC2-scFv-CAR T cells display typical cytokine release only when co-cultured with MUC1* positive cancer cells. huMNC2-scFv-CAR T cells selectively kill MUC1* positive cancers, while stimulating T cell expansion. Conclusions: The relevant target for anti-cancer drugs is the extracellular domain of MUC1*, not full length MUC1. MUC1* is a growth factor receptor that is activated by NME family growth factors. Subtle differences between MUC1* as expressed on healthy stem-like cells and MUC1* expressed on cancerous cells has allowed the development of cancer-specific MUC1* antibodies, especially suited for cancer immunotherapies. Citation Format: Cynthia Carol Bamdad, Andrew K. Stewart, Benoit J. Smagghe, Luke T. Deary, Victoria L. Kohler, Jared L. Dietz. MUC1* targeting CAR T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3330. doi:10.1158/1538-7445.AM2017-3330