Wataru Nunomura

Mapping the Binding Domains Involved in the Interaction between the Plasmodium falciparum Knob-associated Histidine-rich Protein (KAHRP) and the Cytoadherence Ligand P. falciparumErythrocyte Membrane Protein 1 (PfEMP1)

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) clusters at electron-dense knob-like structures on the surface of malaria-infected red blood cells and mediates their adhesion to the vascular endothelium. In parasites lacking knobs, vascular adhesion is less efficient, and infected red cells are not able to immobilize successfully under hemodynamic flow conditions even though PfEMP1 is still present on the exterior of the infected red cell. We examined the interaction between the knob-associated histidine-rich protein (KAHRP), the parasite protein upon which knob formation is dependent, and PfEMP1, and we show evidence of a direct interaction between KAHRP and the cytoplasmic region of PfEMP1 (VARC). We have identified three fragments of KAHRP which bind VARC. Two of these KAHRP fragments (K1A and K2A) interact with VARC with binding affinities (K D(kin)) of 1 × 10−7 m and 3.3 × 10−6 m respectively, values comparable to those reported previously for protein-protein interactions in normal and infected red cells. Further experiments localized the high affinity binding regions of KAHRP to the 63-residue histidine-rich and 70-residue 5′ repeats. Deletion of these two regions from the KAHRP fragments abolished their ability to bind to VARC. Identification of the critical domains involved in interaction between KAHRP and PfEMP1 may aid development of new therapies to prevent serious complications of P. falciparum malaria.

REGULATION OF CD44-PROTEIN 4.1 INTERACTION BY CA2+ AND CALMODULIN : IMPLICATIONS FOR MODULATION OF CD44-ANKYRIN INTERACTION

Erythrocyte membrane skeletal protein 4.1 isoforms have been identified in a variety of non-erythroid cells. However, interactions between protein 4.1 and its binding partners in non-erythroid cell membranes are poorly understood. In the erythrocyte membrane, protein 4.1 binds to the cytoplasmic domain of band 3 and, through this interaction, modulates ankyrin binding to band 3. The sequences LRRRY or IRRRY in band 3 mediate the interaction between band 3 and protein 4.1. The cytoplasmic domain of CD44, a transmembrane glycoprotein found in erythroid as well as non-erythroid cells, has internal sequences SRRRC and QKKKL. We wanted to determine if protein 4.1 binds to CD44 in a fashion analogous to its binding to band 3 and through this interaction modulates ankyrin binding to CD44. We report here that protein 4.1 binds to the cytoplasmic domain of CD44 with a dissociation constant on the order of 10−7 m and that Ca2+ and calmodulin reduce the affinity of this interaction. Furthermore, although independent binding of both protein 4.1 and ankyrin to CD44 could be documented, binding of protein 4.1 prevented subsequent ankyrin binding. These studies have enabled us to identify a potentially important functional role for protein 4.1 in modulating ankyrin binding to CD44.

Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization.

The crystal structure of the core domain (N-terminal 30 kDa domain) of cytoskeletal protein 4.1R has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for either band 3, glycophorin C/D or p55. At a central region of the molecule near where the three lobes are joined are two separate calmodulin (CaM) binding regions. One of these is composed primarily of an α-helix and is Ca 2+ insensitive; the other takes the form of an extended structure and its binding with CaM is dramatically enhanced by the presence of Ca 2+, resulting in the weakening of protein 4.1R binding to its target proteins. This novel architecture, in which the three lobes bind with three membrane associated proteins, and the location of calmodulin binding sites provide insight into how the protein 4.1R core domain interacts with membrane proteins and dynamically regulates cell shape in response to changes in intracellular Ca2+ levels.

Regulation of Protein 4.1R, p55, and Glycophorin C Ternary Complex in Human Erythrocyte Membrane

Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55–4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca2+-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane.

Modulation of Band 3-Ankyrin Interaction by Protein 4.1 FUNCTIONAL IMPLICATIONS IN REGULATION OF ERYTHROCYTE MEMBRANE MECHANICAL PROPERTIES

Protein 4.1 is an important structural component of the erythrocyte membrane. In contrast to our detailed understanding of the role of protein 4.1 in regulating membrane mechanical properties through modulation of spectrin-actin interaction, very little is known regarding the functional implications of protein 4.1 interaction with band 3. In the present study, we explored the potential role of protein 4.1-band 3 interaction in modulating membrane mechanical properties. Based on recent studies which identified the sequence motif IRRRY in band 3 as the protein 4.1 interacting domain, we studied the functional consequences of specific dissociation of band 3-protein 4.1 interaction by the synthetic peptide IRRRY. We show that protein 4.1 bound to the inside-out vesicles could be dissociated from band 3 but not from glycophorin C by IRRRY. Furthermore, incorporation of IRRRY into resealed ghosts resulted in decreased membrane deformability and increased membrane mechanical stability. The observed alterations in membrane properties appears to result from increased band 3-ankyrin interaction following dissociation of protein 4.1 from band 3. These studies have enabled us to identify an important functional role for band 3-protein 4.1 interaction in modulating erythrocyte membrane properties.

Ca2+-dependent and Ca2+-independent Calmodulin Binding Sites in Erythrocyte Protein 4.1 IMPLICATIONS FOR REGULATION OF PROTEIN 4.1 INTERACTIONS WITH TRANSMEMBRANE PROTEINS

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH2-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca2+-independent binding site (A264KKLWKVCVEHHTFFRL) and a Ca2+-dependent binding site (A181KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca2+-dependent and Ca2+-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca2+, implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca2+ and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca2+/CaM to down-regulate 4.1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca2+ and CaM requires binding of CaM to both Ca2+-independent and Ca2+-dependent sites in protein 4.1.

Regulation of protein 4.1R interactions with membrane proteins by Ca2+ and calmodulin.

Red blood cell protein 4.1 (4.1R) is essential for maintaining erythrocyte shape and controlling membrane mechanical properties, such as deformability and stability. The importance of 4.1R has been demonstrated by the dramatic erythrocyte alterations observed in patients lacking this protein. Indeed, 4.1R null red blood cells adopt an elliptical shape and are characterized by unstable membranes. The key role of 4.1R likely results from multiple protein-protein interactions: lateral interactions with the spectrin/actin network and vertical interactions with the cytoplasmic domain of transmembrane proteins glycophorin C (GPC), Band 3 (anion exchanger 1, AE1), and CD44. 4.1R promotes the formation of a ternary complex with GPC and p55 through its 30 kDa membrane-binding domain. Based on the primary structure of the prototypical 80 kDa isoform of 4.1R, functional domains and sites for binding partners have been identified. The others and we have been focusing on the structure and function of the 30 kDa NH2-terminal domain of 4.1R, which is responsible for 4.1R interaction with the transmembrane proteins described above. A major finding is that Ca2+, in association with calmodulin (CaM), plays a critical role in regulation of the interaction of the 30 kDa domain with its various binding partners. This review is a detailed report of our current knowledge regarding 4.1R, and more specifically, 4.1R 30 kDa domain: its primary structure, functions and modulation by Ca2+ and CaM. Emphasis is given on the relationships between structure and function that we have been able to establish through X-ray crystal structure analysis of the 30 kDa membrane-binding domain in 4.1R. Finally, we give insights into the potential roles of 4.1R in the dynamic organization of the membrane skeleton viewed as a complex system.

Enucleation of human erythroblasts involves non-muscle myosin IIB

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

C-REACTIVE PROTEIN IN EEL : PURIFICATION AND AGGLUTINATING ACTIVITY

C-reactive protein was highly purified from Japanese eel (Anguilla japonica) serum by precipitation with phosphatidyl-choline and Ca2+. On SDS-PAGE, eel C-reactive protein (eCRP) migrated as a single band with a molecular weight of 24,000 under reducing and 23,500 under non-reducing conditions. The molecular weight of native eCRP was estimated to be 120,000 by Sephacryl S-300 gel filtration. The eCRP was detected within the albumin region on immunoelectrophoresis. The eCRP showed an agglutinating activity against Streptococcus pneumoniae in the presence of Ca2+, and the activity was inhibited by 1 mM EDTA or 1 mM phosphorylcholine (PC). The eCRP also agglutinated rabbit red blood cells (RRBC), but not human and five other kinds of red blood cell. The hemagglutinating activity was inhibited by glucosamine or mannose. The eCRP formed a precipitin line with histone, protamine, poly(L-lysine) and poly(L-arginine) in agarose gel. The serum levels of eCRP were distributed in 6.8 ng/ml-5.3 mg/ml, n = 187, the mean value being 834 ng/ml.

Changes in serum concentration and mRNA level of rat C-reactive protein

C-reactive protein (CRP) is a typical acute-phase protein found in many animals. Several stimuli, including IL-6 produced by macrophages localized around the site of tissue injury and/or bacterial infection, induce CRP synthesis in liver. One of the authors (W.N.) reported previously that sex hormones also affect the serum CRP concentration in rats. However, little is known about the process of CRP production, including time-dependent changes in the CRP mRNA level in liver. In the present study, we observed time-dependent changes in the serum concentration of CRP and the CRP mRNA level in liver after the injection of turpentine-oil or estradiol-17 beta in rats (Wistar strain). After turpentine-oil injection, CRP mRNA increased, followed by an increase in the serum CRP concentration, indicating that inflammation enhances transcription of the CRP gene. In contrast, upon estradiol-17 beta administration, the serum CRP concentration decreased without any decline in the CRP mRNA level in liver. The latter finding suggests that rats may have an alternative mechanism for regulating the serum concentration of CRP.