Oligosaccharide structures of immunoglobulin G from two patients with carbohydrate-deficient glycoprotein syndrome

The carbohydrate moiety of immunoglobulin G (IgG) from patients with carbohydrate-deficient glycoprotein (CDG) syndrome was analyzed. Galactosyl species were reduced in the reversed-phase chromatogram of pyridylaminated oligosaccharides as compared with child controls, and the hypogalactosylation was remarkable in a patient with typical manifestations. The abnormality was verified by composition analysis of the hydrolyzed monosaccharides from this patient, but the contents of mannose andN-acetylglucosamine were not reduced. Hypogalactosylation is the characteristic feature of IgG molecules in CDG syndrome, in contrast to the oligosaccharide deficiency of transferrin from the same patients. These findings suggest that the molecular phenotypes of different glycoproteins from patients with CDG syndrome are diverse.     

Determination of glycan structures and molecular masses of the glycovariants of serum transferrin from a patient with carbohydrate deficient syndrome type II

Serum transferrin from a child with carbohydrate deficient syndrome type II was isolated by immunoaffinity chromatography and separated into minor and major fractions by fast protein liquid chromatography. The structure of the glycans released from the major fraction by hydrazinolysis was established by application of methanolysis and 1H-NMR spectroscopy. The results led to the identification of an N-acetyllactosamininic type monosialylated, monoantennary Man(1-3) linked glycan. By electrospray-mass spectrometry analysis, the whole serum transferrin was separated into at least seven species (I to VII) with molecular masses ranging from 77 958 to 79 130 Da. On the basis of a polypeptide chain molecular mass of 75 143 Da, it was calculated that the major transferrin species III (78 247 Da) contains two monosialylated monoantennary glycans. The molecular mass of transferrin species V and VI (78 678 and 78 971 Da) suggests that one of their two glycans contains an additional N-acetyllactosamine and a sialylated N-acetyllactosamine units, respectively. Transferrin species I and V were found to correspond to the desialylated forms of species III and VI. The abnormal glycan structures can be explained by a defect in the N-acetylglucosaminyltransferase II activity [Charuk et al. (1995) Eur J Biochem 230: 797-805].     

Simultaneous estimation of the association constants of glycoprotein glycoforms to a common protein by capillary electrophoresis

The efficacy of our capillary electrophoresis method for simultaneous estimation of the association constants of glycoprotein glycoforms to a common target protein was demonstrated using ribonuclease and ovalbumin glycoforms as glycoform models and Lens culinaris agglutinin (LCA) as a protein model. The ribonuclease glycoforms were fairly well separated in the absence of LCA at pH 5.8, but the peaks were retarded without any change of separation profile in the presence of LCA, the retardation becoming greater as LCA concentration increased. The estimated values of apparent association constant (K(a)) were at the 10(6)M(-1) level for all the ribonuclease glycoforms, and there was no significant difference among glycoforms. The high-mannose-type N-glycans released from a mixture of ribonuclease glycoforms gave lower values of K(a) at the 10(4)-10(5)M(-1) level to the same protein, and the glycans having a larger number of the mannose residue gave larger K(a) values. These results imply that the glycan moiety in this glycoprotein might contribute to its binding to the protein, but the polypeptide core played the major role. In contrast, ovalbumin glycoforms gave poorly resolved peaks in the absence of LCA, but they were separated into several peaks in the presence of LCA, which were tentatively assigned based on the knowledge of affinity to this lectin, and K(a) values were estimated simultaneously. The estimated K(a) values were smaller than those of the ribonuclease glycoforms, suggesting the major role of the N-glycan moiety. Thus, capillary electrophoresis allowed simultaneous estimation of K(a) values under common conditions using small amounts of glycoform mixtures and proteins without prior isolation and purification. Comparison of the obtained values will provide useful information on the glycan structure-affinity correlation.     

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N- glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ_max, AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.     

Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer

In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.     

Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient''s plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient''s will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.     

Haptoglobin serum concentration is a suitable biomarker to assess the efficacy of a feed additive in pigs

Levels of haptoglobin and Pig-major acute phase protein (MAP) were analysed in animals from a commercial herd receiving or not a diet enriched with an additive. The group receiving the additive exhibited a decrease in haptoglobin after 3 weeks, suggesting that a better health status has been established, together with an improvement in total body weight and average daily gain. In contrast, Pig-MAP does not significantly change under these conditions. Aujeszky live modified vaccination, which is compulsory in Spain, did cause a significant increment in haptoglobin serum concentration although it did not affect Pig-MAP. The response of acute phase proteins to vaccination was similar in both control and additive-treated groups. Interleukins (IL)-1β and IL-6 was below the detection limits in most of the animals. In conclusion, this study shows that haptoglobin serum concentration, but not Pig-MAP, is a good biomarker to monitorize production parameters and for monitoring Aujeszky modified live vaccine in pigs reared under standard commercial conditions.     

Intracellular localization and surface expression of a stage-specific embryonic glycoprotein

The intracellular and cell surface localization of an embryonic glycoprotein antigen (BL) has been investigated in preimplantation mouse embryos using ultrastructural immunocytochemistry. Several interesting points have emerged: (1) BL antigens are exclusively localized subjacent to the plasma membrane in the cortical region of cells, whereas antigens detected by a control antibody against mouse L cells are distributed throughout the embryo. (2) The distribution of BL antigens is polarized beginning with the first cleavage, with expression confined to the cortex underlying the free or apical portions of cells. No antigen is present underlying regions of cell contact. (3) Although embryonic synthesis of BL antigens does not begin until the two-cell stage, BL antigens are observed in unfertilized eggs, a fact verified by immunoblotting.     

昆虫细胞内N-糖基化途径及人源化糖蛋白表达

虽然昆虫杆状病毒表达系统在蛋白表达领域得到了广泛的应用, 但由于不能表达复杂的末端唾液酸化的N-糖链, 使得该系统在生物制药行业的应用受到了很大的限制。通过比较哺乳动物细胞和昆虫细胞内糖基化途径可知, 其起始步骤一致, 之后再发生分化, 主要表现为3方面, 即昆虫细胞内缺乏哺乳动物细胞所具备的N-乙酰葡萄糖氨转移酶II、 半乳糖基转移酶/N-乙酰氨基半乳糖转移酶、α-2,3-唾液酸转移酶和α-2,6-唾液酸转移酶等延长N-糖链的糖基转移酶; 另外, 昆虫细胞内具有能够特异性地将蛋白质末端的N-乙酰氨基葡萄糖残基从GlcNAcMan3GlcNAc(±α3/6-Fuc)GlcNAc上切除的N-乙酰氨基葡萄糖苷酶及核心α-1,3-岩藻糖基转移酶。本文从上述异同出发, 综述了克服昆虫细胞内不能表达人源化糖蛋白这一缺陷所进行的N-糖基化途径的改造研究--主要集中在昆虫细胞内GlcNAcase的抑制和昆虫细胞内GnT2, GalT/ GalNAcT, ST3及ST6等基因的导入等方面, 结果表明经改造的昆虫细胞可表达人源化糖蛋白, 这将极大地拓宽昆虫杆状病毒表达系统的应用领域。本文还探讨了选择特殊细胞系及特殊培养条件以在昆虫细胞内表达唾液酸化蛋白的可行性。     

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

A novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa).     

Cell cycle dependent expression of a glucose regulated cell surface glycoprotein

Summary A cell surface associated “glucose regulated protein” has been described on nontransformed human fibroblasts. To examine the distribution of that protein on human fibroblasts specific antisera were used. The antisera was used in conjunction with indirect immunofluorescence and revealed that the glucose regulated protein was present as fibers on spread cells. Further, the antisera was used in complement mediated cytotoxicity assays to examine cells during specific stages of the mitotic cell cycle. Fibroblasts were synchronized by serum starvation, hydroxyurea inhibition, or colcemid inhibition followed by mitotic selection. The results demonstrated that the glucose regulated protein was maximally displayed during the G₁ phase of the cell cycle and minimally displayed during the S and M phases. Research was supported by contract AG00697 from the National Institutes of Health.     

Developmental changes of myelin-associated glycoprotein in rat brain: study on experimental hyperphenylalaninemia

We examined developmental changes of myelin-associated glycoprotein (MAG), basic protein (BP), abd proteolipid protein (PLP) in central nervous system myelin isolated from experimental hyperphenylalaninemic rats (PKU rats) and controls. Higher amounts of MAG, including high-molecular-weight MAG in myelin, were found in 12- to 21-day-old control rats than in adult rats. MAG in developing myelin was at a maximum in 18-day-old rats and began to decrease in 21-day-old rats, while PLP and BP in developing myelin increased at these developmental stages. The level of high-molecular-weight MAG decreased in myelin prepared from 21-day-old rats. These results suggest that the decreasing high-molecular-weight MAG is important for compaction of myelin in the early stage of myelination. In myelin from 12- to 18-day-old PKU rats, the ratio of each protein such as MAG, PLP, or BP to that of control was about 0.5 at 12 days, and increased to almost 1.0 at 18 days. The myelination seems to be initially delayed but to be close to that of controls in PKU rats about 18 days old.     

Wolf-Hirschhorn syndrome: A case demonstrated by a cytogenetic study

We present a case with a 4p terminal deletion, evidenced in GTG-banded chromosome study. Phenotypic signs described in the classical Wolf-Hirschhorn syndrome were found on clinical examination of our patient.     

儿童激素耐药型肾病综合征合并星形诺卡菌脑脓肿1例

本文报道1例激素耐药型肾病综合征儿童合并星形诺卡菌(Nocardia asteroides,N.asteroides)脑脓肿。患儿,男性,8岁,临床诊断为原发性肾病综合征(激素耐药型),病理诊断为局灶节段性肾小球硬化症(经典型)。肾穿后第4天患儿出现持续高热、抽搐,时有头痛,抗感染、抗凝治疗效果不佳。复查颅脑磁共振成像(magnetic resonance imaging,MRI)提示多发脑脓肿。头颅脓肿液经穿刺后培养显示为星形诺卡菌感染。予以多种抗生素联合糖皮质激素等治疗2个月,患儿体温正常,头痛缓解,脑脓肿范围明显缩小。因此,肾病综合征患儿在应用激素及免疫抑制剂治疗过程中如出现化脓性炎症,常规抗生素疗效差,应积极寻找病原,高度警惕诺卡菌病及其他机会性感染的可能。     

The best strategy of blood transfusion in patients with Rhnull syndrome

Rh_null syndrome is a very rare disease. Patients with this syndrome present with negative serological Rh typing of E, e, C, c, and D antigens. Only one study has previously discussed Rh_null syndrome in Chinese individuals. We experienced two patients with Rh_null syndrome in China, Rh genotypes being CcDEe in the first patient and CCDee in the second patient. The first patient was a pregnant woman (gravida 2, para 1) with a negative red blood cell (RBC) antibody screen test. The second patient was a middle-aged man, transfused with ccdee, ccdEe, and ccdee RBC products, the pre-transfusion specimen was negative and post-transfusion specimen was anti-c,e, respectively. The hemoglobin level continued to increase in the second patient after being transfused with ccdEe RBC products. In the first patient, the result of the antibody screen test was still negative after artificial abortion. In patients with Rh_null syndrome, RBC products that have the same Rh genotype as the patient can be safely transfused.     

The severe acute respiratory syndrome coronavirus 3a is a novel structural protein

The severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses. Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection. Here we present biochemical data to show that 3a is a novel coronavirus structural protein. 3a was detected in virions purified from SARS-CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full-length protein. In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a, a similar cleavage was observed. Furthermore, co-expression of 3a, membrane and envelope proteins using the baculovirus system showed that both full-length and truncated 3a can be assembled into virus-like particles. This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS-CoV encodes a novel coronavirus structural protein.     

Haptoglobin Proved a Prognostic Biomarker in Peripheral Blood of Patients with Personalized Peptide Vaccinations for Advanced Castration-Resistant Prostate Cancer

The fopA gene encoding a fructooligosaccharide-producing β-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other β-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the β-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any β-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-β-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.     

The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type i by capillary zone electrophoresis

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.Abbreviations CDGS Carbohydrate Deficient Glycoprotein Syndrome - CZE Capillary Zone Electrophoresis - hTf human transferrin - gu HPLC glucose units - EOF electroosmotic flow. Nomenclature: for describing oligosaccharide structures: A(1,2,3,4) indicates the number of antennae linked to the t trimannosyl core - G(0–4) indicates the number of terminal galactose residues in the structure - F core fucose - B bisecting N-acetyl glucosamine (GlcNAc) - S sialic acid - Gal galactose; M - Man mannose