Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.     

A mouse immunoglobulin heavy chain deletion mutant: isolation of a cDNA clone and sequence analysis of the mRNA

The mouse cell line IF2 secretes an immunoglobulin heavy chain lacking the CH1 domain. We have isolated and characterised a recombinant plasmid containing cDNA copies of the IF2 mutant mRNA. The cloned sequence extends from the nucleotides coding for amino acid 96 in the variable region through 100 nucleotides of untranslated region at the 3' end. The sequence of the cDNA insert reveals no discontinuity at the variable-hinge region junction, the site of the CH1 deletion. Experiments employing direct priming on the poly(A) tail of the IF2 heavy chain mRNA suggest that the 3' end of the cDNA clone (sequence C-C-C-T-G-C) is also the 3' end of the mRNA.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Characterization of functional domains of human EB1 family proteins

EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.     

Crystal structure of the amino-terminal microtubule-binding domain of end-binding protein 1 (EB1)

The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules. EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation mechanism is poorly understood. We have determined the 1.45-A resolution crystal structure of the amino-terminal domain of EB1, which is essential for microtubule binding, and show that it forms a calponin homology (CH) domain fold that is found in many proteins involved in the actin cytoskeleton. The functional CH domain for actin binding is a tandem pair, whereas EB1 is the first example of a single CH domain that can associate with the microtubule filament. Although our biochemical study shows that microtubule binding of EB1 is electrostatic in part, our mutational analysis suggests that the hydrophobic network, which is partially exposed in our crystal structure, is also important for the association. We propose that, like other actin-binding CH domains, EB1 employs the hydrophobic interaction to bind to microtubules.     

Novel structural insights into F-actin-binding and novel functions of calponin homology domains

Tandem calponin homology (CH) domains are well-known actin filaments (F-actin) binding motifs. There has been a continuous debate about the details of CH domain-actin interaction, mainly because atomic level structures of F-actin are not available. A recent electron microscopy study has considerably advanced our structural understanding of CH domain:F-actin complex. On the contrary, it has recently also been shown that CH domains can bind other macromolecular systems: two CH domains from separate polypeptides Ncd80, Nuf2 can form a microtubule-binding site, as well as tandem CH domains in the EB1 dimer, while the single C-terminal CH domain of alpha-parvin has been observed to bind to a alpha-helical leucin-aspartate rich motif from paxillin.     

Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.     

紫云英根瘤菌质粒功能研究

紫云英根瘤菌CH203含有3条质粒(pRHa,97MI);pRHb,168MD;pRHc,251MD为共生质粒),用带蔗糖敏感基因Tn5-sacB进行菌株质粒消除和质粒缺失突变株筛选,获得一系列突变株。与野生型菌相比,质粒pRHa的丢失导致菌株结无效根瘤,质粒pRHb的丢失使菌株失去共生能力,在TY培养基平板上菌落变得粗糙,失去了脂多糖(LPSI)。质粒pRHc(共生质粒)的丢失显然失去其菌株的共生能力,同时使菌株抗酸性明显减弱。质粒回复能恢复突变株的表现特征和共生能力。此外,紫云英根瘤菌CH205含有5条大小不同的质粒(分子量42MD～230MD),该菌株某些质粒的消除能显著增强菌株的结瘤固氮能力。研究结果也表明除共生质粒外,紫云英根瘤菌其它质粒明显影响菌株的共生效应。     

The immunoglobulin M heavy chain constant region gene of the channel catfish, Ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule.

The immunoglobulin (IgM) heavy chain constant region gene of the channel catfish, Ictalurus punctatus, has been cloned and characterized. The gene contains four constant region domain-encoding exons (CH1 to CH4) expressed in the secreted form of the immunoglobulin, and two exons encoding the transmembrane (TM) domain utilized in the lymphocyte membrane receptor form of the immunoglobulin. The sequence of a cDNA clone encoding the 3' region of the message for the membrane receptor form of the mu chain indicates that the TM1 exon is spliced directly to the CH3 exon, and not into a site within the CH4 exon, as occurs in the mammals, a shark and an amphibian. This unusual pattern of splicing, which produces a membrane heavy chain that is characteristically smaller than the secreted heavy chain, may be common to all teleost fish.     

Site-specific N-glycosylation of chicken serum IgG

Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE.     

The primary structure of mu-chain-disease protein BOT. Peculiar amino-acid sequence of the N-terminal 42 positions

The complete primary structure of the mu heavy-chain disease (mu-HCD) protein BOT has been determined. The monomeric HCD-mu-chain consists of 391 amino-acid residues, lacking the VH and mu CH1 domains but including the entire CH2, CH3 and CH4 domains (349 residues). The sequence of the preceding 42 N-terminal residues which we designate as the "pre-C-part" presents no homology to any known variable or constant immunoglobulin sequence, but contains an internal homology of positions 10-19 to positions 20-29. The origin of the "pre-C-part" structure and the deletion of the mu CH1 domain of protein BOT are discussed.     

Sos-mediated activation of rac1 by p66shc

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1-eps8-e3b1 tricomplex. The NH(2)-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1-eps8-e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.     

Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG

Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3–CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2–CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.     

Molecular cloning of rabbit gamma heavy chain mRNA.

A cDNA library of rabbit spleen mRNA was screened for immunoglobulin heavy chain sequences. In this paper we report the nucleotide sequence of two cDNA clones containing part of the constant region of the rabbit gamma heavy chain mRNA. The sequence encodes part of the CH2 domain (amino acids 268 to 340), the entire CH3 domain (amino acids 341 to 447) and the 3' untranslated region. This nucleotide sequence has been compared to the corresponding sequences of mouse gamma 1, gamma 2a and gamma 2b genes. The homologies between rabbit gamma chain gene sequence and each of the mouse gamma chain gene sequences are of the same magnitude order. This comparison shows that the CH2 domains are more homologous to each other than CH3 domains or 3' untranslated sequences. The presence of species specific nucleotide positions suggests that mouse gamma chain genes could have evolved from a common ancestor shortly after the mouse-rabbit species separation. Genomic blot analysis of rabbit liver DNA with the rabbit C gamma probes shows a limited number of related sequences, with little restriction site polymorphism between individual rabbits.     

Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.     

The CIS Family: Negative Regulators of JAK–STAT Signaling

A family of cytokine-inducible SH2 proteins (CISs) has recently been identified and the members are growing in number. In this family, the central SH2 domain and approximately 40 amino acids at the C-terminus (CIS homology domain; CH domain) are well conserved, while the N-terminal region shares little similarity and varies in length. Most CISs appear to be induced by several cytokines and at least three of them (CIS1, CIS3 and JAB) negatively regulate cytokine signal transduction. Forced expression of CIS1 inhibits STAT5 activation by binding of CIS1 to cytokine receptors, and CIS3 and JAB directly bind to the kinase domain of JAKs, thereby inhibiting kinase activity. Therefore, these CIS family members seem to be present in a classical negative feedback loop of cytokine signaling. They may also play a role in the mutual suppression of cytokine actions frequently found in immune and inflammatory responses. Precise molecular mechanisms of the signal inhibition and their physiological functions will be addressed in the near future. The CH domain is also found in several interesting genes containing WD-40 repeats, SPRY domains, ankyrin repeats, and GTPases. However, the function of the CH domain remains to be determined.     

Sites in the CH3 domain of human IgA1 that influence sensitivity to bacterial IgA1 proteases

The influence of regions, other than the hinge, on the susceptibility of human IgA1 to cleavage by diverse bacterial IgA1 proteases, was examined using IgA1 mutants bearing amino acid deletions, substitutions, and domain swaps. IgA1 lacking the tailpiece retained its susceptibility to cleavage by all of the IgA1 proteases. The domain swap molecule alpha1alpha2gamma3, in which the CH3 domain of IgA1 was exchanged for that of human IgG1, was resistant to cleavage with the type 1 and 2 serine IgA1 proteases of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae, but remained sensitive to cleavage with the metallo-IgA1 proteases of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus mitis. Substitution of the IgA1 Calpha3 domain motif Pro440 -Phe443 into the corresponding position in the Cgamma3 domain of alpha1alpha2gamma3 resulted now in sensitivity to the type 2 IgA1 protease of N. meningitidis, indicating the possible requirement of these amino acids for sensitivity to this protease. For the H. influenzae type 2 protease, resistance of an IgA1 mutant in which the CH3 domain residues 399-409 were exchanged with those from IgG1, but sensitivity of mutant HuBovalpha3 in which the Calpha3 domain of bovine IgA replaces that of human IgA1, suggests that CH3 domain residues Glu403, Gln406, and Thr409 influence sensitivity to this enzyme. Hence, unlike the situation with the metallo-IgA1 proteases of Streptococcus spp., the sensitivity of human IgA1 to cleavage with the serine IgA1 proteases of Neisseria and Haemophilus involves their binding to different sites specifically in the CH3 domain.