Regulation of glycan structures in animal tissues: transcript profiling of glycan-related genes

Abstract

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.

FIGURE 1.

Comparison of microarray and qRT-PCR data for glycan-related transcripts from mouse liver and kidney. RMA values from the GLYCOv2 gene chip microarray analysis are plotted against relative transcript abundance data from qRT-PCR analysis (normalized to Rpl4) for 149 glycan-related genes (plotted on a log₁₀ scale on both axes). Data obtained from mouse liver (upper panel) and kidney (lower panel) are shown. Transcripts determined as present in both methods (see “Experimental Procedures”) are shown as filled circles. Transcripts that were called as absent in the microarray data set are shown as open circles, and transcripts that were not detected by either method are shown as triangles. The correlation coefficient (solid line) for transcripts scored as present in both analyses were R² = 0.39 for liver (upper panel) and R² = 0.24 for kidney (lower panel). The dashed horizontal line represents a best-fit line for the transcripts called as absent in the microarray analysis indicating a lower limit of detection in this method. The dotted vertical line represents the lower limit of detection for qRT-PCR analysis.

FIGURE 2.

Relative transcript abundance for genes involved in N-glycan lipid-linked oligosaccharide precursor biosynthesis. A, schematic representation of the N-glycan lipid-linked oligosaccharide biosynthetic pathway was adapted from Ref. (including a key for glycan and lipid structural components in the pathway). Linkages are shown for each step of the biosynthetic pathway, and the numbers in the blue ovals designate the pathway steps in A that link to the transcript abundance data in the corresponding numbered step in B (plotted as a histogram on a log₁₀ scale). Relative transcript abundances for the four mouse tissues are presented as a clustered set of histograms above the corresponding pathway step number (blue numbered oval) and gene names. Multiple genes for a given pathway step are listed in cases where multiple distinct subunits contribute to catalysis or where several genes within a common family encode enzymes capable of creating the specified linkage.

FIGURE 3.

Relative transcript abundance for processing steps involving N-glycan trimming and branching in the endoplasmic reticulum and Golgi complex. A shows the schematic representation for N-glycan trimming, branching, and modifications for high mannose, hybrid, and complex N-linked oligosaccharides using the glycan schematic nomenclature indicated in the legend. Labeling of pathway steps in the schematic diagram and the corresponding steps in B are as described in Fig. 2. Transcript abundance values were determined by qRT-PCR and plotted on a log₁₀ scale as a clustered set of histograms for the respective adult mouse tissues above each pathway step as described in Fig. 2. Multiple genes for a given pathway step are listed in cases where several genes encode enzymes capable of creating the specified linkage or where the substrate specificity of multiple members of a given gene family have not been sufficiently defined to make a restricted enzyme assignment.

FIGURE 4.

Relative transcript abundance for processing steps leading to the elaboration of complex capping reactions for N-glycan, O-glycan, and glycolipid structures in mouse tissues. Upper panel shows the schematic representation for complex capping reactions for N-glycan, glycolipid, and O-glycan classes indicated by the boxed structures in the left of the panel. Structures sub-terminal to the nonreducing terminal GlcNAc residues indicated by asterisks in the boxed structures are subsequently depicted as “R” in the remainder of the figure. The branched scheme for glycan processing to various complex termini are indicated using the glycan schematic nomenclature indicated in the legend. Colored backgrounds for individual branches of the complex processing pathways are labeled as indicated to designate different classes of complex termini. Labeling of pathway steps in the upper schematic diagram using blue numbered ovals also correspond with the designated steps in the lower panel histogram as described in Fig. 2. Transcript abundance values for the individual pathway steps were determined by qRT-PCR and plotted on a log₁₀ scale as a clustered set of histograms for the respective adult mouse tissues above each pathway step as described in Fig. 2. Multiple genes for a given pathway step are listed in cases where several genes encode enzymes capable of creating the specified linkage or where the substrate specificity of multiple members of a given gene family has not been sufficiently defined to make a restricted enzyme assignment.

FIGURE 5.

Relative abundance of N-glycan structures in adult mouse tissues determined by MALDI-MS analysis. N-Glycan structural data were previously obtained from animal tissue extracts (14) following enzymatic release, permethylation, and MALDI-MS approaches by the Consortium for Functional Glycomics. Raw spectral data were analyzed as described under “Experimental Procedures” to generate lists of mean-centered ion clusters for each glycan mass followed by integration of the corresponding ion currents and manual assignment of glycan structures. Putative glycan structures contributing less than 1.5% to the total ion current integral of all assigned glycan structures are not shown. Ion current integrals were normalized and plotted as a percentage of the total ion current for the given data set. Schematics for identified carbohydrates are indicated above each set of data from mouse kidney (black bars), liver (dark gray bars), testis (light gray bars), and brain (white bars). In some cases, more than one structure was identified for the same mass, and schematics depicting these structures are indicated by the first letter of the tissue where it was identified.

FIGURE 6.

Correlations of relative transcript abundance for glycan-related genes with corresponding N-glycan structures in mouse tissues. The relative abundances of various classes of N-glycan structures (left panels) were obtained by the normalized integration of MALDI-MS ion current data as described in Fig. 5. The percentage of total N-glycan structures containing core α1,6-Fuc residues (A), nonreducing terminal (“peripheral”) α1,3-Fuc (C), bisecting β1,4GlcNAc on the N-glycan trimannosyl core (E), and the relative percentage of oligomannose versus hybrid and complex structures (G) in mouse kidney (K), liver (L), testis (T), and brain (B) are indicated. Corresponding relative transcript abundances determined by qRT-PCR (right panels) are plotted on a log₁₀ scale for gene(s) responsible for the addition of those respective residues, Fut8 (B), Fut4, Fut7, Fut9,Fut10, and Fut11 (D), Mgat3 (F), Mgat1 (H), and the Golgi α-mannosidases, (GMIA (Man1a1), GMIB (Man1a2), and GMIC (Man1c1)) (I) are shown. For all panels the tissue sources for the histogram abundance data are designated as follows: mouse kidney (K, black bars), liver (L, dark gray bars), testis (T, light gray bars), and brain (B, white bars). Error bars in the relative transcript abundance data represent the mean ± 1 S.D.

FIGURE 7.

Relative transcript abundance for sialyltransferases and neuraminidases in mouse tissues. Sialyltransferases are grouped according to the type of linkage they create upon transfer from a CMP-sialic acid donor and respective gene names for the sialyltransferases and neuraminidases are listed at the bottom of the histogram. Relative transcript abundance data are plotted as a clustered set of histograms for the respective genes as indicated for kidney (black bars), liver (dark gray bars), testis (light gray bars), and brain (white bars). Error bars represent the mean ± 1 S.D.