Patterns of protein glycosylation in bovine placentomes as a function of gestational age and in retained versus non-retained placenta.

The formation of placenta at the beginning of pregnancy and its separation at parturition require not only deep remodelling of extracellular matrix, which mainly consists of proteins conjugated with sugar moieties, but also the cooperation with cells from both maternal and foetal parts of placenta. The aim of the study was to compare the patterns of selected conjugated proteins with sugar moieties between pregnant and term placenta as well as between released and retained placenta in cows. Placental samples from healthy pregnant cows (3-5 months of pregnancy) were collected at a slaughterhouse (n = 6), and parturient samples were collected during caesarean section at term and retrospectively divided into retained (n = 6) and released (n = 6). The pattern of selected sugar moieties conjugated with proteins was detected by use of lectin blotting with Phaseolus Vulgaris leucoagglutinin, Maackia Amurensis and Sambucus Nigra (Elderberry). The comparison and analysis of obtained band patterns showed differences between their number, molecular weight and abundance related to the intensity of staining. Samples from 3 to 4 months showed similarities, while at the 5th month, clear differences were visible in all 3 lectins, which were used in this study. Samples from retained/released placenta expressed significant differences in PHA-L and SNA pattern in the foetal part. Obtained results indicate that the development of placenta related to extracellular matrix and accompanying cells from both sides of placenta shows dynamic changes during pregnancy. Moreover, in the case of animals with the retention of foetal membranes the patterns of proteins conjugated with sugar moieties are altered, suggesting that the changes in extracellular matrix metabolism can be involved in the attachment and detachment of the placenta in cows.

Lectin Blotting of Cell Lysates.

Glycans are composed of branched structures consisting of monosaccharides, such as glucose and galactose linked by glycosidic bonds. Glycans are often bound to proteins and lipids and are localized at the cell surface. They are deeply involved in a wide range of multicellular systems inside and outside the cells, such as the quality control of glycoproteins, cell-cell communication, and various diseases. While western blotting uses antibodies to detect proteins, lectin blotting uses lectins, which are glycan-binding proteins, to detect glycans on glycoconjugates, such as glycoproteins. Lectin blotting was first reported in the early 1980s and has been widely used in life science for several decades. However, it is not straightforward to obtain consistent data using lectin blotting, which tends to show high backgrounds and lab-to-lab variation. Here, we describe the protocol used in our laboratory for lectin blotting following protein separation by SDS-PAGE to detect glycoproteins extracted from cell membrane fractions. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Extraction and quantification of proteins from cell lysate Basic Protocol 2: Lectin peroxidase labeling and lectin blotting.

Rapid glycosylation analysis of mouse serum glycoproteins separated by supported molecular matrix electrophoresis.

Previously, we developed a novel separation technique, namely, supported molecular matrix electrophoresis (SMME), which separates mucins on a PVDF membrane that impregnated with a hydrophilic polymer (such as polyvinyl alcohol), so it has the characteristics that are compatible with glycan analysis of the separated bands. Here, we describe the first instance of the application of SMME to mouse sera fractionation and demonstrate their differences from the pooled human sera fractionation by SMME. Furthermore, we have developed a fixation method for the lectin blotting of SMME-separated glycoproteins by immersing the SMME membranes into acetone solvent followed by heating. It showed that the amount of protein samples required for SMME were reduced more than 4-fold than that of the process of SDS-PAGE. We applied these techniques for the detection of glycosylation patterns of serum proteins from Fut8+/+ and Fut8-/- mice, further analyzed N-linked and O-linked glycans from the separated γ-bands by mass spectrometry, and demonstrated that there are α2,8-sialylated O-glycans contained in mouse sera glycoproteins. SMME can provide simple, rapid sera fractionation, glycan profiling differences between the bands of two samples and a new insight into the underlying mechanism that responsible for related diseases. SIGNIFICANCE: We describe that the first application of SMME can separate mouse serum proteins into six bands and identify the major protein components of each fraction in mouse serum separated by SMME. Furthermore, we successfully developed a fixation method for lectin blotting of SMME-separated glycoproteins and applied to the detection of glycosylation patterns of serum glycoproteins from Fut8+/+ and Fut8-/- mice, also, the method is promising for detecting glycan profiling differences between two samples in both research and clinical settings.

Glycan Recognition and Application of P-Type Lectins.

Cation-dependent mannose 6-phosphate receptor (CD-MPR) and cation-independent MPR (CI-MPR) belong to the P-type lectin family. Both intracellular and cell surface MPRs can recognize and bind with the terminal mannose 6-phospahte (M6P) residues of N-glycans attached to the mammalian lysosomal enzymes and the related co-factors. Domain9 (Dom9), which is one of the extracytoplasmic region of the CI-MPR, has relatively higher affinity for M6P residues. Here we describe the production of recombinant Dom9-His protein by Pichia pastris, purification, and application as a probe for lectin blotting.

The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs.

The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin - MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra.

Monosaccharide profiling of silkworm (Bombyx mori L.) nervous system during development and aging.

Glycoconjugates have various functions in differentiation, development, aging and in all aspects of normal functioning of organisms. The reason for increased research on this topic is that glycoconjugates locate mostly on the cell surface and play crucial biological roles in the nervous system including brain development, synaptic plasticity, learning, and memory. Considering their roles in the nervous system, information about their existence in the insect nervous system is rather sparse. Therefore, in order to detect monosaccharide content of N- and O-glycans, we carried out capLC-ESI-MS/MS analysis to determine the concentration changes of glucose, mannose, galactose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, xylose, arabinose, and ribose monosaccharides in the nervous system of Bombyx mori during development and aging processes. In addition to LC-MS, lectin blotting was done to detect quantitative changes in N- and O-glycans. Developmental stages were selected as 3rd (the youngest sample), 5th (young) larval instar, motionless prepupa (the oldest sample), and pupa (adult development). Derivatization of monosaccharides was performed with a solution of PMP agent and analyzed with capLC-ESI-MS/MS. For lectin blotting, determination of glycan types was carried out with Galanthus nivalis agglutinin and Peanut agglutinin lectins. In all stages, the most abundant monosaccharide was glucose. Although all monosaccharides were present most abundantly in the youngest stage (3rd instar), they are generally reduced gradually during the aging process. It was observed that amounts of monosaccharides increased again in the pupa stage. According to lectin blotting, N- and O-linked glycoproteins expressions were different and there were some specific glycoprotein expression differences between stages. These findings suggest that the glycosylation state of proteins in the nervous system changes during development and aging in insects in a similar fashion to that reported for vertebrates.