Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.     

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III₂; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.     

Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk

The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.     

O-linked glycosylation in maize-expressed human IgA1

O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis. Approximately 90% of hIgA1 was modified in this way. An average molar ratio of six Ara units per molecule of hIgA1 was revealed. Substantial sequence similarity was identified between the HC hinge region of hIgA1 and regions of maize extensin-family of hydroxyproline-rich glycoproteins (HRGP). We propose that because of this sequence similarity, the HC hinge region of maize-expressed hIgA1 can become a substrate for posttranslational conversion of Pro to Hyp by maize prolyl-hydroxylase(s) with the subsequent arabinosylation of the Hyp residues by Hyp-glycosyltransferase(s) in the Golgi apparatus in maize endosperm tissue. The observation of up to six Pro/Hyp hydroxylations combined with extensive arabinosylation in the hIgA1 HC hinge region is well in agreement with the Pro/Hyp hydroxylation model and the Hyp contiguity hypothesis suggested earlier in literature for plant HRGP. For the first time, the extensin-like Hyp/Pro conversion and O-linked arabinosylation are described for a recombinant therapeutic protein expressed in transgenic plants. Our findings are of significance to the field of plant biotechnology and biopharmaceutical industry-developing transgenic plants as a platform for the production of recombinant therapeutic proteins.     

Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1

The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.     

Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS)

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto alpha-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

Asymptotic Expansions and Estimation of the Expected Error Rate for Equal-Mean Discrimination with Uniform Covariance Structure

The asymptotic error rate of the equal-mean, uniform-covariance-matrix classification rule is approximated by a first order asymptotic expansion. The approximation is compared for accuracy with a Monte Carlo simulation. Finally, an estimator of the error rate and an estimator of the variance of the error rate estimator are derived and applied to a classical example.     

Telocytes within human skeletal muscle stem cell niche

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.