Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: Characterization of an N -glycosylation mutant Pro − 5 Chinese Hamster Ovary (CHO) cell line generated by employment of the CRISPR/Cas9 method. The coding sequence (CDS) of the Mgat2 gene from 1 to 42 is shown for the parental cell line, Pro − 5, and the isolated clonal cell line, K16 ( A ); K16 has the Mgat2 gene silenced due to insertion of c after the 22nd nucleotide, as denoted by the red arrow. The red font indicates the changes in the codon as a result of the insertion; Representative flow cytometry histograms of five fluorescently labelled lectins binding to each of the CHO cell lines ( B ); Ratios of mean fluorescence values of the N -glycosylation mutant cell lines to the parental cell line were determined and compared from four separate experiments ( n = 4) ( C ). Mean ratio values close to 1 indicate that the binding of the N -glycosylation mutants were similar to Pro − 5. (*, p < 0.02) denote that K16 was significantly different from Lec1.

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Glycoproteomics, Mutagenesis, Generated, CRISPR, Sequencing, Isolation, Flow Cytometry, Binding Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: Lectin blots of whole cell lysates from parental and N -glycosylation mutant CHO cell lines. Pro − 5, K16, and Lec1 samples were probed with Galanthus nivalis Lectin (GNL) and Phaseolus vulgaris Leucoagglutinin (L-PHA), as indicated ( A ); SDS gels with similar levels of protein were evaluated by coomassie blue staining ( B ). Plus signs signify the cell line examined. Lines adjacent to blot and gel denote molecular weight standards in kDa: 250, 150, 100, 75, 50, 37, 25 and 20 from top to bottom.

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Glycoproteomics, Mutagenesis, Staining, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: Type of N -glycan influences cell–cell adhesion. Microscopy images were acquired for Pro − 5, K16, and Lec1 cell lines ( A ); Particles (>5 cells/cluster) examined are encircled in black. The histogram represents the area of cell clusters from about 200 images for each cell line, as indicated ( B ), AU, arbitrary unit.

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Glycoproteomics, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: Electrophoretic migration of E-cadherin expressed in the various CHO cell lines. Western blots of E-cadherin glycoprotein in total membranes from transfected Pro − 5, K16, and Lec1, as denoted by the plus sign ( A ); dashed line on blot was utilized to illustrate small electrophoretic shifts for the three immunobands. Total membranes of E-cadherin transfected K16 cell line were digested (+) and undigested (−) with PNGase F, Endo H, and neuraminidase (neu) ( B ); dashed arrow indicates glycosylated E-cadherin and solid line represents the unglycosylated form. Solid lines below and above immunobands were employed to emphasize the small electrophoretic shifts. The numbers adjacent to the Western blots represent the Kaleidoscope markers (in kDa).

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Western Blot, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: Predominant expression of hybrid N -glycans impacts the localization of E-cadherin at the cell–cell border in a different manner than predominantly expressed complex or oligomannose N -glycans. Microscopy images were acquired in TIRF ( upper panels ), wide-field ( middle panes ), and DIC ( lower panels ) modes for EGFP tagged E-cadherin expressed in K16 cell line ( A ). To directly compare the localization of E-cadherin to the cell–cell interface with complex and oligomannose N -glycans, TIRF ( upper panels ) and DIC ( lower panels ) images were also acquired for E-cadherin transfected Pro − 5 and Lec1 cell lines ( B ); Representative scale bar (5 µM) was same for all images. Cell–cell interface is denoted by white arrows. Fluorescence intensity measurements were ascertained at the cell–cell interface (I cell–cell ), and away from the cell–cell interface (I cell ) of the cell membrane patch to determine the amount of E-cadherin at cell–cell border relative to that away from the cell–cell border (I cell–cell /I cell ). The bar on the graph reports the I cell–cell /I cell mean value of E-cadherin expressed in the K16 cell line while the lines designated as complex and oligomannose are the mean values for E-cadherin expressed in Pro − 5 and Lec1 cell lines, respectively ( C ); The ratio of I cell–cell /I cell value for a given N -glycan type to complex type of N -glycan illustrates differences in the spatial arrangement of E-cadherin expressed with different predominant N -glycan types ( D ). At the p < 0.01 level, the differences of the population means are significantly different by one-way ANOVA with Bonferroni adjustment (*).

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Microscopy, Transfection, Fluorescence, Membrane, Glycoproteomics

Journal: International Journal of Molecular Sciences

Article Title: Predominant Expression of Hybrid N -Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N -Glycans

doi: 10.3390/ijms17060925

Figure Lengend Snippet: E-cadherin modified cell–cell adhesion in cells expressing different types of N -glycans. Microscopy images were acquired for E-cadherin transfected Pro − 5 and non-transfected Pro − 5, K16, and Lec1 ( A ); particles (>5 cells per cell cluster) analyzed are encircled. Mean values of the area ( B ), and number ( C ) of cell clusters were plotted on bar graphs from four separate days of experiments, as indicated. n values were greater than 199 fields and 267 clusters. Black asterisks denote significant differences in mean values of a non-transfected cell line, and E-cadherin transfected cell line at a probability of p < 0.00001 using student t -test ( B ). Gray asterisks represent significant differences in mean values of non-transfected or E-cadherin transfected CHO cell lines at a probability of p < 0.05 using one-way ANOVA with Bonferroni adjustments. NS signifies mean values were not significantly different.

Article Snippet: Parental Pro − 5 and glycosylation mutant Pro − Lec1 (Lec1) CHO cells were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Modification, Expressing, Microscopy, Transfection