Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Crystal structure of muconolactone isomerase at 3.3 A resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Genic mutation rates in mammals: local similarity,chromosomal heterogeneity,and X-versus-autosome disparity

The reduction of mutation rates on the mammalian X chromosome relative to autosomes is most often explained in the literature as evidence of male-driven evolution. This hypothesis attributes lowered mutation rates on the X chromosome to the fact that this chromosome spends less time in the germline of males than in the germline of females. In contrast to this majority view, two articles argued that the patterns of mutation rates across chromosomes are inconsistent with male-driven evolution. One article reported a 40% reduction in synonymous substitution rates (Ks) for X-linked genes relative to autosomes in the mouse-rat lineage. The authors argued that this reduction is too dramatic to be explained by male-driven evolution and concluded that selection has systematically reduced mutation rate on the X chromosome to a level optimal for this male-hemizygous chromosome. More recently, a second article found that chromosomal mutation rates in both the human-mouse and mouse-rat lineages were so heterogeneous that the X chromosome was not an outlier. Here again, the authors argued that this is at odds with male-driven evolution and suggested that selection has modulated chromosomal mutation rates to locally optimal levels, thus extending the argument of the first mentioned article to include autosomes. Here, we reexamine these conclusions using mouse-rat and human-mouse coding-region data. We find a more modest reduction of Ks on the X chromosome, but our results contradict the finding that the X chromosome is not distinct from autosomes. Multiple statistical tests show that Ks rates on the X chromosome differ systematically from the autosomes in both lineages. We conclude that the moderate reduction of mutation rate on the X chromosome of both lineages is consistent with male-driven evolution; however, the large variance in mutation rates across chromosomes suggests that mutation rates are affected by additional factors besides male-driven evolution. Investigation of mutation rates by synteny reveals that synteny blocks, rather than entire chromosomes, might represent the unit of mutation rate variation.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.