Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia

We have analyzed the hemoglobins of a young German patient with β-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the β-globin through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the $\text{G}\text{T}\text{G}\text{→}\text{G}\text{G}\text{G}$ mutation at codon 126 leading to the synthesis of Hb Dhoburi or α₂β₂126(H₄)Val→Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G→C) mutation that leads to a rather severe β⁺-thalassemia and the $\text{G}\text{TG}\text{→}\text{A}\text{TG}$ mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered β-chain variant, named Hb Baden, was present for only 2–3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G→C) thalassemic mutation. The chromosome with the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) and the IVS-I-5 (G→C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G→C) mutation living in different countries failed to detect the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) change.     

Studies on the stereoselective synthesis of a protected α-D-Gal-(1→2)-D-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-D-Gal-(1→2)-D-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-D-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-D-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH(3) at C-1 and benzoyl groups at C-4 and C-6.     

Conformational analysis of a Chlamydia-specific disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl in aqueous solution and bound to a monoclonal antibody: Observation of intermolecular transfer NOEs

The disaccharide -Kdo-(28)--Kdo (Kdo: 3-deoxy-d-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide -Kdo-(28)--Kdo-(2O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all ¹H NMR signals of -Kdo-(28)--Kdo-(2O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that -Kdo-(28)--Kdo-(2O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to -Kdo-(28)--Kdo-(2O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody–disaccharide complex.     

Hemoglobin M Iwate is caused by a C→T transition in codon 87 of the human α1-globin gene

Summary DNA restriction, molecular cloning, and sequencing methods have been used to characterize the mutation leading to the methemoglobinemia HbM Iwate. It could be demonstrated that the HbM Iwate defect is caused by a point mutation involving a transition from C to T in the first position of codon 87 of the ₁-globin gene. Furthermore, the HbM Iwate mutation can directly be identified upon RsaI digestion. This direct detection of the mutation on the gene level is of significant advantage for differential diagnostic purposes.     

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.     

CST-II's recognition domain for acceptor substrates in α-(2→8)-sialylations

CST-II is a bacterial sialyltransferase known for its ability to perform α-(2→8)-sialylations using GM(3) related trisaccharide substrates. Previously, we probed the enzyme's substrate specificity and developed an efficient synthesis for α-(2→8)-oligosialosides, and we suggested that CST-II could have a very small substrate recognition domain. Here we report our full studies on CST-II's recognition feature for acceptor substrates. The current study further demonstrates the versatility of CST-II in preparing complex oligosaccharides that contain α-(2→8)-oligosialyl moieties.     

Synonymous codon usage bias and overexpression of a synthetic gene encoding Interferon α2b in yeast

To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut⁺ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×10⁵ IU/mL. Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)     

Structure of a complex asparagine bound glycopeptide from horse pancreatic ribonuclease containing (2→3) and (2→6) linked sialic acid residues

The primary structure of the main glycopeptide obtained by pronase digestion of horse pancreatic ribonuclease has been investigated by 360 MHz ¹H-NMR spectroscopy and methylation analysis. The results demonstrate that this glycopeptide has the following structure:

This is the first time that the presence of both (2→3) and (2→6) linked sialic acid residues in a glycopeptide has been demonstrated.     

Synthese von O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)-(2→4)-O-(3-desoxy-α-d-manno-2-octulopyranosylonsäure)- (2→6)-O-(2-amino-2-desoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-desoxy-d-glucopyranose

Benzyl-3-O-benzyl-2-benzyloxycarbonylamino-6-O-[2-benzyloxycarbonyl-amino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)- β-d-glucopyranosyl]-2-deoxy-α-d-glucopyranoside was coupled with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl bromide)onate (13) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′-O-tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O-[methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-α-d-manno-2-octulopyranosyl)onate]-(2→4)-O-{methyl [3-deoxy-7,8-O-(tetraisopropyldisiloxane-1,3-diyl)-α-d-manno-2-octulopyranosyl]-onate}-(2→6)-O- [2-benzyloxycarbonylamino-2-deoxy-3,4-O-(tetraisopropyldisiloxane-1,3-diyl)-β-d-glucopyranosyl]- (1→6)-3-O-benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α-d-glucopyranoside. A series of deblocking steps gave O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)-(2→4)-O-(3-deoxy-α-d-manno-2-octulopyranosylonic acid)- (2→6)-O-(2-amino-2-deoxy-β-d-glucopyranosyl)-(1→6)-2-amino-2-deoxy-d-glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.     

Hemoglobin Stanleyville II (α78 Asn→Lys) is associated with a 3.7-kb α-globin gene deletion

Summary A family with one homozygote and three heterozygotes for hemoglobin Stanleyville II (78 AsnLys) has been analyzed by -globin gene mapping. The pattern of restriction fragments with the enzymes BamHI and BglII demonstrated that the globin variant is associated with a 3.7-kb -globin gene deletion.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Syntheses of α- and β-Glycosyl Donors with a Disaccharide β-D-Gal-(1→3)-D-GalNAc Backbone

The synthesis of thioglycoside glycosyl donors with a disaccharide -D-Gal-(1 3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-- and 1-thio--D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1 4), rather than (1 3)-linked, disaccharide product formed. The aglycon transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio--D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azido group in position 2 of galactosamine and can hence be used to form -glycoside bond. Azido group and the aglycon nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the -glycosyl donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM₁.     

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

Summary A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a CT transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Lipoprotein-associated phospholipase A(2) interacts with phospholipid vesicles via a surface-disposed hydrophobic α-helix

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis, and sepsis. Lp-PLA(2) also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA(2) plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA(2) with dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.     

Synthesis and biological activity of α-galactosyl ceramide KRN7000 and galactosyl (α1→2) galactosyl ceramide

We herein report a faster and less cumbersome synthesis of the biologically attractive, α-galactosyl ceramide (α-GalCer), known as KRN7000, and its analogues. More importantly, the use of a silicon tethered intramolecular glycosylation reaction gave easy access to the diglycosyl ceramide Gal(α1→2)GalCer, which has been shown to require uptake and processing to the biologically active α-GalCer derivative.     

Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.