| 1. |
- Atanasova, Diana, 1991-, et al.
(författare)
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Glycoproteomic profile of human tissue-nonspecific alkaline phosphatase expressed in osteoblasts
- 2024
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Ingår i: JBMR Plus. - : Oxford University Press. - 2473-4039. ; 8:2
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Tidskriftsartikel (refereegranskat)abstract
- Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate and ATP to provide inorganic phosphate, thus controlling the inorganic pyrophosphate/inorganic phosphate ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has 5 potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303, and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl+/− expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and liquid chromatography with tandem mass spectrometry. The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all 5 sites of TNALP, as well as core fucosylation on 4 out of 5 sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.
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| 2. |
- Gawria, Ghassaan, et al.
(författare)
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A comparison of stability of chemical analytes in plasma from the BD Vacutainer (R) Barricor (TM) tube with mechanical separator versus tubes containing gel separator
- 2020
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Ingår i: Journal of clinical laboratory analysis (Print). - : WILEY. - 0887-8013 .- 1098-2825. ; 34:2
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a need of prolonged stability of certain chemical analytes in lithium heparin tubes with separators. A new tube with a mechanical separator has recently been launched (Barricor (TM)), which according to the manufacturer may have these benefits. The aim of this study was to evaluate stability performance of this tube in comparison with plasma gel tubes under clinically realistic circumstances. Methods: Blood was collected in tubes containing lithium heparin with different separators; gel separator (Vacutainer (R) PST (TM), Becton Dickinson and Vacuette (R), Greiner bio-one) and mechanical separator (Vacutainer (R) Barricor (TM), Becton Dickinson). All tubes had an aspiration volume of 3 mL and were centrifuged at similar time and force. Tubes were transported manually or by car. Seven analytes from 122 patients were analyzed after 3 to 80 hours by Cobas c701 (Roche). Results The Barricor (TM) tube showed increased stability of phosphate and potassium and similar stability of aspartate aminotransferase, glucose, homocysteine, lactate dehydrogenase, and magnesium compared with gel tubes. Maximal allowable bias for phosphate was exceeded after 68 hours for Barricor (TM) tubes compared with 29 or 35 hours for gel tubes and for potassium after 40 hours for Barricor (TM) tubes vs 9 or 12 hours for gel tubes. Transportation did not affect stability. Hemolysis index was slightly lower in Barricor tubes than in gel tubes (P = .01). Conclusion Implementing the new Barricor (TM) tube will improve stability of potassium and phosphate in plasma. Blood sampling facilities far from the laboratory may benefit from using these tubes, thus diminishing preanalytical errors.
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| 3. |
- Landberg, Eva, 1966-, et al.
(författare)
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Changes in Glycosylation of Human Bile-Salt-Stimulated Lipase during Lactation
- 2000
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 377:2, s. 246-254
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Tidskriftsartikel (refereegranskat)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption–ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.
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| 4. |
- Landberg, Eva, 1966-, et al.
(författare)
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Detection of molecular variants of prolactin in human serum, evaluation of a method based on ultrafiltration
- 2007
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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 376:1-2, s. 220-225
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIn human blood, there are several molecular variants of prolactin with different biological effects. There is a need for new methods to detect and quantify these variants in order to fully understand the pathophysiological role of prolactin.MethodsA method based on ultrafiltration was optimized, validated and compared to PEG precipitation. Serum samples from 84 patients were analyzed before and after pre treatment on two immunoassays, Elecsys (Roche) and Access (Beckman). Protein G precipitation was used to confirm presence of macroprolactin.ResultsThe recovery of prolactin after ultrafiltration was lower than after PEG precipitation. A limit of 40% recovery after PEG precipitation corresponded to 27% recovery after ultrafiltration. Using these limits there were total agreement regarding detection of macroprolactin (rs = 0.96). In contrast, recovery of prolactin in samples without macroprolactin showed a considerable disagreement between ultrafiltration and PEG precipitation (rs = 0.48). Within-run CV was 4% for the ultrafiltration method. The correlation coefficient (r) between the immunoassays was 0.96 after ultrafiltration.ConclusionsUltrafiltration can be used to compare different prolactin immunoassays and to detect macroprolactin in assays with interference from PEG. For samples without macroprolactin ultrafiltration may give additional information reflecting individual variations of other molecular variants of prolactin.
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| 5. |
- Landberg, Eva, 1966-
(författare)
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Free oligosaccharides and glycosylation of bile salt-stimulated lipase in human milk
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bile salt-stimulated lipase (BSSL) is a glycosylated protein present in milk at a concentration of 100-200 mg/L. It is an enzyme important for fat digestion in the newborn infant. The protein backbone contains one possible site for N-glyeosylation and several sites for 0-glyeosylation. The glycosylation of BSSL may be important for protection against proteolytic degradation and/or secretion of BSSL. The oligosaeeharides bound to BSSL may also, together with other protein-bound oligosacchatides and free oligosaecharides in milk, play an important role in the defense against pathogenic microorganisms. Human milk contains approximately 5-20 g/L of free milk oligosacchatides, and more than 100 different structures have been identified. There are individual differences in the content of milk oligosaeeharides depending on Lewis and seeretor status.Milk samples were collected from healthy donors at different times during lactation. BSSL was purified from the milk of five donors. Structural characterization of BSSL glyeosylation was performed by high-performance anionexchange chromatography (HP AEC), Bio-Gel P-4 chromatography, lectin affinity chromatography, gas chromatography coupled to mass-spectrometry (MS) and mattix assisted laser desorption-ionization time-of-flight MS. Certain carbohydrate epitopes were detected by monoclonal antibodies and lectins. Some of the methods above were used in combination with ptior derivatization, desialylation or digestion with different exo- and endoglycosidases. Thirteen major free oligosaccharides were quantified in milk from five individuals. Free milk oligosacchatides were purified by P4-Gel chromatography and analyzed by HPAEC.HP AEC coupled to pulsed amperometric detection is extensively used for analysis and quantification of oligosaccharides. Separation is achieved using highly alkaline conditions that lead to ionization of some of the hydroxyl groups, which can then interact with the anion-exchange matrix. The effect of colunm temperature was examined in a range of 13 to 40 oC. A large variation in retention times was found depending on small differences in colunm temperature. Moreover, individual oligosaccharides did not show the same temperature dependence. By use of different column temperatures, HP AEC could be optimized for analysis of milk oligosaccharides.BSSL was found to contain approximately one N-linked and nine O-linked oligosaccharides. The 0-glycans were stmcturally heterogeneous and contained· fucose and/or sialic acid. Each 0-glycan contained an average of eight monosaccharide units. The major N-linked oligosaccharides on human BSSL were mono-sialylated biantennary complex type structures with or without one, two or three fucose residues.Recombinant human BSSL expressed in CHO and C-127 cells were analyzed and found to be differently glycosylated than native BSSL. In contrast to native BSSL, recombinant BSSL did not contain fucose. On BSSL expressed in C-127 cells, the O-glycans were shorter and more extensively sialylated than O-glycans on native BSSL. The majority of N-linked oligosaccharides on recombinant BSSL had the same core structure (biantennary complex type) as native BSSL.Glycosylation of BSSL changed during lactation. BSSL had a higher carbohydrate and sialic acid content in the first lactation month. There was also a shift from preferentially α2-6 to α2-3 linked sialic acid on the protein-bound oligosaccharides during lactation. This shift was also found for free sialylated milk oligosaccharides, and suggests a change in the activity of certain sialyltransferases during lactation.A gradual increase in the expression of the fucosylated carbohydrate epitope Lewis x (Galß1-4[Fucal-3]GlcNAc-) was found on BSSL during the whole lactation period. This was reflected in a higher relative amount of fucosylated N-linked oligosaccharides present on BSSL later in lactation. A similar increase in fucosylation was indicated by analysis of free milk oligosaccharides. One of the major milk oligosaccharides, 3-fucosyllactose (3-FL), also increased in concentration during lactation. However, lacto-N-fucopentaose (LNFIII), the only free milk oligosaccharide containing the Lewis x epitope, showed a constant concentration. This finding does not exclude the possibility that the same fucosyltransferase is involved in the synthesis of Lewis x on BSSL, 3-FL and LNFIII. The precursor of LNFIII, lacto-N-neotetraose (LNnT) showed a marked decrease during lactation, which may explain the different pattem found for LNFIIl. The increase of 3-FL and Lewis x on BSSL was found for all individuals. The other free oligosaccharides studied decreased during lactation, except for lacto-N-fucopentaose li (LNFII), lacto-di-fucotetraose (LDFT) and 3-sialyllactose (3-SL), which showed constant concentrations.Total fucosyltransferase activity decreased during lactation in milk from both secretors and non-secretors. The specific α1-3 fucosyl transferase activity toward lactose also decreased during lactation, which indicated that fucosyltransferase activity in milk does not reflect the activity in the mammary epithelial cells.In conclusion, there are changes in glycosylation during lactation, which involves both protein-bound and free milk oligosaccharides. The different patterns for individual oligosaccharides indicate both down and up regulation of certain glycosyltransferases in the mammary gland during lactation. The importance of these changes for the infant's adaptation to the environment remains to be elucidated.
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