Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice

Abstract

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcbeta1-4 branch synthesis on the Manalpha1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcbeta1-4 branch on the Manalpha1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity.

Fig. 1.

Tissue distribution pattern and mutagenesis of the Mgat4b-encoded GnT-IVb glycosyltransferase. (A) Expression profile of mouse Mgat4b RNA transcripts among total RNA samples from indicated tissues. The bottom panel represents the ethidium bromide-stained gel as a loading control of RNA samples. (B) Mouse genomic clone of Mgat4b bearing exons 2, 3, and 4 (black boxes) used for construction of the targeting vector with the pflox plasmid as indicated. Restriction enzyme sites are indicated: (AL) Apa LI; (Bg) Bgl II; (Bm) Bam HI; (E) Eco RI; (Nh) Nhe I; (S) Sal I; (Sp) Spe I; (Sh) Sph I; (Xb) Xba I; (Xh) Xho I. (C) Homologous recombination produces the Mgat4b F[tkneo] allele. Following Cre recombination and Gancyclovir selection, embryonic stem cell clones are isolated bearing the type 1 (Δ, deleted) and type 2 (F, floxed) alleles. Represented restriction enzyme sites are same as those in B. (D) Southern blot analysis of ES cell genomic DNA probed with a genomic or a loxP probe confirmed the predicted allelic structures. Allelic structures in parental R1 ES cell and derived ES cell clones (3-6D, 12A, 1E4, and 1A) are indicated as the position of the individual band on the blot with a genomic probe and the number of the loxP site on the blot with the loxP probe. (E) Adult mice genotypes with germline modifications to the Mgat4b gene, including Mgat4bF and Mgat4bΔ alleles. Mouse genotype is indicated as the position of the band on the blot with the genomic probe: A, wild type; B, Mgat4bΔ homozygote; C, Mgat4bΔ heterozygote; D, Mgat4bF heterozygote.

Fig. 2.

Aberrant Mgat4a expression in GnT-IVb deficiency. Total RNAs obtained from mouse tissues of wild-type and Mgat4b null were subjected to electrophoresis on formaldehyde denaturing gel, and then blotted onto the nylon membrane. Mgat4a transcripts were detected by probing with Mgat4a-specific cDNA fragment. Each bottom panel represents the ethidium bromide-stained gel image that indicates similar loading of analyzed RNA. Ethidium bromide-stained reference gels represent the quality of RNA samples.

Fig. 3.

MALDI-TOF mass spectra of permethylated N-glycans derived from mouse pancreas. Glycomic profiles of wild-type (A and B upper panels) and GnT-IVa/-IVb double deficient (A and B lower panels) pancreas were obtained from the 50% MeCN fraction from a C₁₈ Sep-Pak (Material and methods). For clarity major ions are shown. Cartoon structures are according to the Consortium for Functional Glycomics (http://www.functionalglycomics.org) guidelines. All molecular ions are [M+-Na]^+-. Putative structures based on composition, tandem mass spectrometry, and the literature are shown. Structures that show sugars outside a bracket have not been unequivocally defined. The signals at m/z 4623, 5072, 5521, 5970, and 6419, designated with bold characters, correspond to structure at m/z 4174 extended with LacNAc repeats as indicated in the corresponding signals. The signals at m/z 4653, 5102, 5551, 6000, and 6449, designated with bold italic characters, correspond to structure at m/z 4204 extended with LacNAc repeats as indicated in the corresponding signals.

Fig. 4.

MALDI-TOF-TOF spectra of the [M+-Na]^+- molecular ions from mouse pancreas. Signals present at m/z 3755 of wild-type and GnT-IVa/-IVb double deficiency in Figure 3 were subjected to tandem MS and the results are shown in (A) and (B), respectively. Structures labeled with “M” and “m” indicate major and minor abundances, respectively. The horizontal arrows on the spectra indicate losses from the molecular ion [M+-Na]^+- of the designated N-glycan sequences in insets. (A) In the wild-type pancreas, the ion m/z 690 established the presence of Galα1-3Gal epitope, while the ions at m/z 3292, 3088, and 2639 corresponded to losses of HexHexNAc, Hex₂HexNAc, and Hex₃HexNAc₂ respectively. Moreover, the signal at m/z 3303 corresponded to the loss of FucHexNAc from the reducing side of the glycan, indicative of core fucosylation. (B) In the GnT-IVa/-IVb double deficient pancreas, the spectrum was characterized by substantial differences suggesting structural alterations; compare the ratios of the ions at m/z 2639 and 3088 between the wild-type and GnT-IVa/-IVb double deficiency pancreas. Signals present at m/z 3695 (wild-type) and 3693 (GnT-IVa/-IVb double deficiency) in Figure 3 were zoomed in (C) and (E), respectively. Structures that show sugars outside a bracket have not been unequivocally defined. Those ions were subjected to tandem MS and the results are represented in (D) and (F), respectively. MALDI-TOF-TOF MS/MS of the above two ions established the presence of a fucosylated terminated antenna (m/z 660), of a Galα1-3Gal epitope (m/z 690), and of terminated NeuGc antenna (m/z 877). (D) In the wild-type mouse pancreas, the spectrum was characterized by dominant fragment ions at m/z 2839, 3028, and 3058 corresponding to losses of HexHexNAcNeuGc, Hex₂HexNAc, and FucHexHexNAc respectively. The fragment ions at m/z 3232 and 3288 corresponded to losses of HexHexNAc and NeuGc respectively. The above data taken together indicated the presence of three different structures as indicated in the inset of Figure 4D. (F) In the GnT-IVa/-IVb double deficient pancreas, the spectrum was characterized by losses (m/z 1983, 2433, 2839, and 3288) and fragment ions (m/z 877) corresponding mainly to the structure indicated in Figure 4F.