Truncated, InactiveN-Acetylglucosaminyltransferase III (GlcNAc-TIII) Induces Neurological and Other Traits Absent in Mice That Lack GlcNAc-TIII
2002; Elsevier BV; Volume: 277; Issue: 29 Linguagem: Inglês
10.1074/jbc.m202276200
ISSN1083-351X
AutoresRiddhi Bhattacharyya, Mantu Bhaumik, T. Shantha Raju, Pamela Stanley,
Tópico(s)Galectins and Cancer Biology
ResumoN-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complexN-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3 Δ) or for a targeted mutation in theMgat3 gene (previously called Mgat3 neo , but herein renamed Mgat3 T37 because the allele generates inactive GlcNAc-TIII of ∼37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutralN-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3 T37/T37 homozygotes in a mixed or 129SvJ background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned byMgat3 T37/T37 mothers were rescued by wild-type foster mothers. None of these Mgat3 T37/T37 traits were exhibited by Mgat3 Δ/Δ mice or by heterozygous mice carrying the Mgat3 T37 mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3 T37 and Mgat3 Δmutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed inMgat3 T37/T37 brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse. N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complexN-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3 Δ) or for a targeted mutation in theMgat3 gene (previously called Mgat3 neo , but herein renamed Mgat3 T37 because the allele generates inactive GlcNAc-TIII of ∼37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutralN-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3 T37/T37 homozygotes in a mixed or 129SvJ background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned byMgat3 T37/T37 mothers were rescued by wild-type foster mothers. None of these Mgat3 T37/T37 traits were exhibited by Mgat3 Δ/Δ mice or by heterozygous mice carrying the Mgat3 T37 mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3 T37 and Mgat3 Δmutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed inMgat3 T37/T37 brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse. N-acetylglucosaminyltransferase Chinese hamster ovary erythroagglutinin from P. vulgaris phosphate-buffered saline matrix-assisted laser desorption/ionization time-of-flight mass spectrometry The N-glycans of mammalian glycoproteins vary widely in structure, but the biological significance of this variation is largely unknown. A well studied example is the bisecting GlcNAc. This residue is transferred to the β-linked Man of the core ofN-glycans by the glycosyltransferase termedN-acetylglucosaminyltransferase III (GlcNAc-TIII1; EC2.4.1.144) (1Narasimhan S. J. Biol. Chem. 1982; 257: 10235-10242Abstract Full Text PDF PubMed Google Scholar), the product of the Mgat3 gene (2Ihara Y. Nishikawa A. Tohma T. Soejima H. Niikawa N. Taniguchi N. J. Biochem. (Tokyo). 1993; 113: 692-698Crossref PubMed Scopus (106) Google Scholar). The presence of the bisecting GlcNAc alters the lectin binding properties of a cell, a fact initially revealed by the gain-of-function Chinese hamster ovary (CHO) glycosylation mutant LEC10, which expresses GlcNAc-TIII (3Campbell C. Stanley P. J. Biol. Chem. 1984; 259: 13370-13378Abstract Full Text PDF PubMed Google Scholar). LEC10 cells are ∼15-fold more resistant to ricin and ∼10-fold more sensitive to the toxicity of the erythroagglutinin fromPhaseolus vulgaris (E-PHA) compared with wild-type CHO cells, reflecting dramatic changes in binding of these lectins toN-linked Gal residues of cell-surface glycoproteins. Similarly, the ectopic expression of an Mgat3cDNA reduces the expression of terminal α3-Gal residues, a key determinant in xenotransplantation (4Miyagawa S. Murakami H. Takahagi Y. Nakai R. Yamada M. Murase A. Koyota S. Koma M. Matsunami K. Fukuta D. Fujimura T. Shigehisa T. Okabe M. Nagashima H. Shirakura R. Taniguchi N. J. Biol. Chem. 2001; 276: 39310-39319Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Koyota S. Ikeda Y. Miyagawa S. Ihara H. Koma M. Honke K. Shirakura R. Taniguchi N. J. Biol. Chem. 2001; 276: 32867-32874Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The regulated expression of the Mgat3 gene could therefore control the binding of animal lectins like galectins to cell-surface N-glycans with a bisecting GlcNAc and thereby modulate cellular interactions. The expression of the Mgat3 gene varies in a tissue-specific fashion in mice (6Bhaumik M. Seldin M.F. Stanley P. Gene (Amst.). 1995; 164: 295-300Crossref PubMed Scopus (39) Google Scholar, 7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar) and has been correlated with the onset of hepatoma in rats (8Nishikawa A. Fujii S. Sugiyama T. Hayashi N. Taniguchi N. Biochem. Biophys. Res. 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Chem. 1997; 272: 2866-2872Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and overexpression of GlcNAc-TIII modulates several cellular properties, including the metastasis of B16 melanoma cells (15Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8754-8758Crossref PubMed Scopus (260) Google Scholar), the sensitivity of K562 cells to killing by natural killer cells and enhanced spleen colonization (16Yoshimura M. Ihara Y. Ohnishi A. Ijuhin N. Nishiura T. Kanakura Y. Matsuzawa Y. Taniguchi N. Cancer Res. 1996; 56: 412-418PubMed Google Scholar), and the binding of epidermal growth factor to the epidermal growth factor receptor (17Rebbaa A. Yamamoto H. Saito T. Meuillet E. Kim P. Kersey D.S. Bremer E.G. Taniguchi N. Moskal J.R. J. Biol. Chem. 1997; 272: 9275-9279Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Transgenic mice overexpressing GlcNAc-TIII in liver exhibit altered secretion of certain glycoproteins (18Ihara Y. Yoshimura M. Miyoshi E. Nishikawa A. Sultan A.S. Toyosawa S. Ohnishi A. Suzuki M. Yamamura K. Ijuhin N. Taniguchi N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2526-2530Crossref PubMed Scopus (48) Google Scholar), but no change in the development of diethylnitrosamine-induced hepatocarcinogenesis (19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). In bone marrow, overexpression of GlcNAc-TIII suppresses stroma-dependent hematopoiesis (20Yoshimura M. Ihara Y. Nishiura T. Okajima Y. Ogawa M. Yoshida H. Suzuki M. Yamamura K. Kanakura Y. Matsuzawa Y. Taniguchi N. Biochem. J. 1998; 331: 733-742Crossref PubMed Scopus (13) Google Scholar). To identify new functions for N-glycans with a bisecting GlcNAc, the Mgat3 gene has been inactivated by targeted mutations in the mouse. Priatel et al. (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar) used a Cre/loxP strategy to delete the Mgat3 gene coding region, whereas Bhaumik et al. (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar) inserted apgkneo cassette into the Mgat3 gene to disrupt the coding region. Both mutations retard the progression of liver tumors in homozygotes treated with diethylnitrosamine and phenobarbital (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar, 19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). Although deletion of the Mgat3 gene coding region (Mgat3 Δ mutation) causes no overt phenotypic traits in a mixed or C57BL/6 genetic background (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar), elimination of GlcNAc-TIII activity by disruption of the Mgat3 gene was preliminarily reported to give rise to neurological and other consequences in mice of mixed genetic background (21Bhaumik M. Sundaram S. Stanley P. Glycobiology. 1996; 6: 720Google Scholar). We now describe these traits in detail and show that they are maintained or become more severe after backcrossing 11 generations onto the 129SvJbackground. By contrast, Mgat3 Δ/Δ mice do not exhibit the same traits in either mixed or inbred genetic backgrounds. The targeted Mgat3 allele gives rise to a truncated, enzymatically inactive GlcNAc-TIII that induces a partial mutant phenotype in Mgat3 T37 /Δcompound heterozygotes. Humans with related nonsense mutations in theMGAT3 gene may have neurological or behavioral problems. UDP-[6-3H]Gal (10 Ci/mmol), UDP-[6-3H]GlcNAc (41.60 Ci/mmol), and concanavalin A-Sepharose were from Amersham Biosciences. Biotinylated E-PHA lectins were from Vector Labs, Inc. Bio-Gel P-2 (45–95 mesh), the DC protein assay reagent, and AG 1-X4 resin (200–400 mesh, Cl− form) were from Bio-Rad. Pronase (Streptomyces griseus) and EDTA-free protease inhibitor tablets were from Roche Molecular Biochemicals. Triton X-100 and bovine serum albumin (fraction V) was from Sigma. G418, fetal bovine serum, and α-medium were from Invitrogen. Ecolume was from ICN Biomedicals, andN-glycanase from New England Biolabs Inc. The Mgat3 tm1Pst insertion mutation was previously generated by introducing apgkneo gene in reverse orientation into the NcoI site of the Mgat3 gene in WW6 embryonic stem cells and termed Mgat3 neo (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar, 19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). In this study, we show that a truncated GlcNAc-TIII of ∼37 kDa (predicted 371 or 374 amino acids based on encoded stop codons) is produced from the targeted allele. Henceforth, this mutant allele will therefore be termed Mgat3 T37 to better reflect the nature of its product. Heterozygous Mgat3 T37 /+ progeny from chimeras generated by injection of C57BL/6 blastocysts withMgat3 T37 /+ WW6 embryonic stem cells were crossed to CD1 mice, and cousins were interbred to obtain F1, F2, and F3 generations ofMgat3 +/+,Mgat3 T37 /+, andMgat3T37/T37 mice. The Mgat3 tm1Jxm mutation (termed Mgat3 Δ) was previously generated by deleting the coding region of the Mgat3 gene using a Cre/loxP strategy (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar).Mgat3 Δ/+ mice in a CD1 mixed genetic background were generated from clone 18Mgat3 Δ/+ R1 embryonic stem cells (provided by Dr. Jamey D. Marth) (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar) by injection into C57BL/6 blastocysts; crossing of chimeras to CD1 mice; and interbreeding cousins to obtain F1, F2, and F3 Mgat3 +/+, Mgat3 Δ/+, andMgat3 Δ/Δ mice as described (19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). To obtain compound heterozygotes, Mgat3 T37/T37 mice were crossed to Mgat3 Δ/Δ mice to generateMgat3 T37 /Δ mice.Mgat3 T37 /+ mice from the CD1 mixed genetic background were backcrossed to wild-type 129SvJmice for 11 generations, and heterozygous progeny were interbred to derive mice heterozygous and homozygous for theMgat3 T37 mutation in a 129SvJbackground. Methods for detecting wild-type Mgat3 and mutantMgat3 T37 and Mgat3 Δ alleles by PCR and Southern analysis of tail genomic DNA using the primers and probes shown in Fig. 1 A have been previously described (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar,12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar, 19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). Total RNA from mouse kidney or brain was prepared using Trizol reagent (Invitrogen), electrophoresed on a denaturing 1.2% agarose gel, transferred to membrane, and probed with a 584-bpBglI/PstI fragment of the Mgat3 gene coding exon (probe V; see Fig. 1 A) or the neogene probe (probe X; see Fig. 1 A) as described (6Bhaumik M. Seldin M.F. Stanley P. Gene (Amst.). 1995; 164: 295-300Crossref PubMed Scopus (39) Google Scholar, 12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar). Total RNA from embryonic head at different times of gestation was isolated using Trizol reagent and then electrophoresed and probed with the Mgat3 gene coding exon probe as described above. To determine that the ∼6.5-kb transcript from the targeted allele was generated from endogenous Mgat3 gene promoter(s), reverse transcription was performed in 20 μl containing 10 μg of total RNA from Mgat3 T37/T37 kidney, 75 mm KCl, 3 mm MgCl2, 25 mm Tris-HCl (pH 8.3), 0.5 mm dNTPs, 10 mm dithiothreitol, 1000 units/ml RNase inhibitor (Promega), 0.5 μg of oligo[dT(n)6] oligonucleotides (Amersham Biosciences), and 200 units of Superscript II reverse transcriptase (Invitrogen). After incubation at 42 °C for 1 h, a 5-μl aliquot was subjected to PCR using primers 63 and 99 (see Fig.1 A). PCR was performed through 40 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min, followed by a final extension step at 72 °C for 15 min. Products of the size expected (0.4 kb) for transcripts from the sense Mgat3 T37 gene orientation were obtained. Parental CHO cells and the gain-of-function CHO mutant LEC10 (3Campbell C. Stanley P. J. Biol. Chem. 1984; 259: 13370-13378Abstract Full Text PDF PubMed Google Scholar) were cultured in suspension at 37 °C in complete α-medium containing 10% fetal bovine serum. cDNAs encoding the Mgat3 gene coding region or theMgat3 T37 gene were cloned into the pcDNA3.1 vector (Invitrogen). Plasmid DNA (5 μg) was linearized byXhoI digestion, purified by ethanol precipitation, and transfected into parental CHO or LEC10 cells (∼4 × 106 in 750 μl of phosphate-buffered saline (PBS)) using a Bio-Rad electroporator (pulse, 0.3 kV; capacitance, 975 microfarads; and average time constant, 12 ms). Selection of stable transfectants was performed by adding G418 (1 mg/ml active weight) 24 h after plating. Colonies that survived G418 selection were isolated and characterized. GlcNAc-TIII and GlcNAc-TI activities were assayed in a volume of 40 μl as described (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar) using GlcNAc-terminating biantennary glycopeptide at pH 6.5 (GlcNAc-TIII) or Man5-GlcNAc2-Asn at pH 6.25 (GlcNAc-TI). UDP[3H]GlcNAc (Amersham Biosciences) was diluted to a specific activity of ∼10,000 cpm/nmol, and ∼24 nmol were included in each assay. β4Galactosyltransferase activities were assayed using GlcNAc as acceptor and UDP[3H]Gal as donor as described (22Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For lectin blots, CHO cells or kidney or brain extracts (50 μg of protein) from mice perfused with cold PBS (pH 7.2) were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane. Desialylation was performed in 25 mm sulfuric acid at 80 °C for 1 h. Biotinylated lectins (Vector Labs, Inc.) were used to detect the presence of the bisecting GlcNAc onN-glycans as described (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar, 19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). For Western blots, a rabbit polyclonal antibody against amino acids 120–136 of mouse GlcNAc-TIII (TRMLEKPSPGRTEEKTE), synthesized by the Laboratory for Macromolecular Analysis and Proteomics at the Albert Einstein College of Medicine, was used as described (19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar). To obtain kidneyN-glycans for analysis, mice were perfused with PBS, and kidney pieces at 150 mg/ml were homogenized for 5 min on ice in buffer containing 10 mm Tris-HCl (pH 7.4), 250 mmsucrose, 0.25 mg/ml leupeptin, 0.7 mg/ml pepstatin, 0.28 mg/ml aprotinin, and 0.5 mg/ml phenylmethylsulfonyl fluoride. Triton X-100 was added to 2% by volume; nuclei were removed by centrifugation; glycerol was added to 20% by volume; and extracts were stored at –70 °C. Protein concentration was measured with the Bio-Rad protein dye reagent. N-Glycans were released from glycoproteins immobilized on polyvinylidene difluoride membranes by treatment withN-glycanase as described (22Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Mass spectrometry was performed on a Voyager DE Biospectrometry Workstation (PerSeptive Biosystems) equipped with delayed extraction as described previously (22Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). A nitrogen laser irradiated samples at 337 nm, and an average of 240 scans were taken. The instrument was operated in linear configuration (1.2-m flight path), and an acceleration voltage of 20 kV was used after a 60-ns delay. Samples (0.5 μl) were applied to a polished stainless steel target to which 0.3 μl of matrix was added and dried under vacuum (50 × 10−3 torr). Oligosaccharide standards were used to achieve a two-point external calibration for mass assignment of ions (23Papac D.I. Wong A. Jones A.J. Anal. Chem. 1996; 68: 3215-3223Crossref PubMed Scopus (257) Google Scholar, 24Raju T.S. Briggs J.B. Borge S.M. Jones A.J. Glycobiology. 2000; 10: 477-486Crossref PubMed Scopus (391) Google Scholar). 2,5-Dihydroxybenzoic acid and 5-methoxysalicylic acid matrix was used for neutral oligosaccharides. Mice were maintained in a barrier facility with food and water provided ad libitum and weighed on the same day at the same time each week. To measure gait, each hind paw was dipped in Indian ink, and the mouse was allowed to walk undisturbed on a 4.5 × 3.5-foot piece of Whatman filter paper. The distance between hind footsteps was measured by the manual method as described (25Steinberg H. Sykes E.A. McBride A. Terry P. Robinson K. Tillotson H. J. Pharmacol. Methods. 1989; 21: 103-113Crossref PubMed Scopus (10) Google Scholar). To record the altered leg clasp reflex, each mouse was suspended by the tail for a maximum of 2 min and photographed using a video camera. The time of holding of the rear leg clasp was recorded. Brain sections prepared by cryostat from perfused mice were thawed at room temperature for 30 min, fixed in 4% paraformaldehyde and PBS at room temperature for 15 min, and washed three times with PBS. The sections were acetylated twice in 0.1m triethanolamine and acetic anhydride at room temperature for 10 min and washed twice with PBS, followed by dehydration in 30, 50, 70, 90, 95, and 100% ethanol for 5 min each. After dipping in chloroform for 5 min at room temperature and in 0.2× SSC for 1–2 min, prehybridization was carried out in a moist chamber with 50% formamide in 5× SSC at 45 °C for 3 h. For hybridization, 35S-UTP-labeled antisense and sense RNAs encoding the Mgat3 gene coding region were prepared from a mouse Mgat3 cDNA (6Bhaumik M. Seldin M.F. Stanley P. Gene (Amst.). 1995; 164: 295-300Crossref PubMed Scopus (39) Google Scholar) using T7 or SP6 RNA polymerase (Promega); probes were hydrolyzed; and hybridization, washing, and autoradiography were performed as described previously (26Yang J. Bhaumik M. Liu Y. Stanley P. Glycobiology. 1994; 4: 703-712Crossref PubMed Scopus (18) Google Scholar). Previous studies showed that the insertion of a pgkneo gene in reverse orientation into the NcoI site of theMgat3 gene abrogates GlcNAc-TIII activity from mouse tissues (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar). However, the stop codons that occur in the pgkneoreverse sequence (Fig. 1 A) did not cause nonsense-mediated decay, as was obtained previously with a similarly disrupted Mgat1 gene coding exon (27Ioffe E. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 728-732Crossref PubMed Scopus (363) Google Scholar, 28Ioffe E. Liu Y. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11041-11046Crossref PubMed Scopus (32) Google Scholar). Northern analysis (Fig. 1 B) and reverse transcription-PCR (data not shown) revealed read-through transcripts fromMgat3 gene promoter(s) that were present at similar or greater levels than transcripts from the endogenous Mgat3gene in brain and kidney. The size of the novel transcripts was ∼6.5 kb and included the ∼4.7-kb Mgat3 gene coding and untranslated regions with the ∼1.8-kb antisense transcript from the pgkneo cassette. Transcription was fromMgat3 gene promoter(s) because reverse transcription-PCR of kidney and brain transcripts from homozygous mutant mice using an oligo(dT) primer for reverse transcription and primers 63 and 99 (Fig.1 A) for PCR gave the predicted product (data not shown), and Western analysis (see below) revealed a protein product. Sense transcripts of ∼1.8 kb that would encode an active neomycin phosphotransferase protein were not detected by Northern analysis in kidney (Fig. 1 B) or brain (data not shown) RNA. The signal present in all lanes at ∼1.8 kb in Fig. 1 B presumably comes from cross-hybridization of the probe with 18 S RNA because it was present in wild-type mice lacking the pgkneo gene. A truncated GlcNAc-TIII predicted to comprise 371 or 374 amino acids was produced from the ∼6.5-kb transcripts. It was detected by Western analysis of mouse tissues, faintly in LEC10 CHO cells, and readily in CHO transfectants using a peptide affinity-purified anti-GlcNAc-TIII antibody made against N-terminal amino acids 120–136 (Fig.1 C). The disrupted Mgat3 gene that gives rise to the truncated product will henceforth be referred to as the mutantMgat3 T37 allele. Truncated GlcNAc-TIII was also present in brain extracts from Mgat3 T37/T37 mice (data not shown). Wild-type GlcNAc-TIII migrated at a molecular mass of ∼70 kDa, just above a nonspecific band detected in kidney and brain. GlcNAc-TIII assays of stable CHO transfectants overexpressing wild-typeMgat3 cDNA gave an activity of 17.9 nmol/mg/h compared with <0.01 nmol/mg/h for cells expressing truncated GlcNAc-TIII in the same vector. Both types of transfectant expressed equivalent activities of unrelated glycosyltransferases such as β4galactosyltransferase and GlcNAc-TI (data not shown). Previous studies detected no GlcNAc-TIII activity in kidney or brain extracts ofMgat3 T37/T37 mice (12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar). Mgat3 Δ/Δ mice from predominantly C57BL/6 or 129SvJ genetic backgrounds exhibit no overt phenotype (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar), 2J. D. Marth, personal communication.whereas Mgat3 T37/T37 mice on a mixed genetic background were observed, in a preliminary report, to possess several distinctive traits different from those of wild-type littermates (21Bhaumik M. Sundaram S. Stanley P. Glycobiology. 1996; 6: 720Google Scholar). To investigate the phenotype associated with theMgat3 T37 mutation and to determine whether the discrepancy with the Mgat3 Δ mutation was due to genetic background, Mgat3 Δ/+ andMgat3 T37 /+ mice were generated on a similar mixed background (predominantly 129SvJ and CD1) (19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar) and bred through cousin matings to obtain wild-type, heterozygous, and homozygous progeny for phenotypic comparisons. The lack of complexN-glycans with a bisecting GlcNAc was confirmed in homozygotes by analyzing the N-glycans released byN-glycanase from kidney glycoproteins ofMgat3 T37/T37 , Mgat3 Δ/Δ, and wild-type littermates by MALDI-TOF-MS (Fig.2). Spectra from two wild-type and two homozygous mutants of each strain were distinguished only by the presence (in wild-type) and absence (in mutants) of glycans with masses predicted for bi-, tri-, or tetraantennary N-glycans with an unsubstituted HexNAc residue (TableI). Based on previous lectin blot analyses (7Priatel J.J. Sarkar M. Schachter H. Marth J.D. Glycobiology. 1997; 7: 45-56Crossref PubMed Scopus (95) Google Scholar, 12Bhaumik M. Harris T. Sundaram S. Johnson L. Guttenplan J. Rogler C. Stanley P. Cancer Res. 1998; 58: 2881-2887PubMed Google Scholar, 19Yang X. Bhaumik M. Bhattacharyya R. Gong S. Rogler C.E. Stanley P. Cancer Res. 2000; 60: 3313-3319PubMed Google Scholar) and reports on similar analyses of kidneyN-glycans from other mice (29Chui D. Sellakumar G. Green R. Sutton-Smith M. McQuistan T. Marek K. Morris H. Dell A. Marth J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1142-1147Crossref PubMed Scopus (185) Google Scholar, 30Wang Y. Tan J. Sutton-Smith M. Ditto D. Panico M. Campbell R.M. Varki N.M. Long J.M. Jaeken J. Levinson S.R. Wynshaw-Boris A. Morris H.R., Le, D. Dell A. Schachter H. Marth J.D. Glycobiology. 2001; 11: 1051-1070Crossref PubMed Scopus (123) Google Scholar), this “extra” HexNAc residue present only in wild-type N-glycans represents the bisecting GlcNAc. The spectra show a remarkable reproducibility in the profile of N-glycans synthesized by the kidneys of unrelated mice from the two strains (Table I). It is important to note that the presence of C-terminally truncated GlcNAc-TIII inMgat3 T37/T37 kidney did not significantly change the spectrum of complex N-glycans expressed byMgat3 T37/T37 compared withMgat3 Δ/Δ mice.Table INeutral N-glycans from kidneys of Mgat3 T37/T37 and Mgat3 Δ/Δ MicePredicted neutral, complex N-glycan structurePredicted mass ([M + Na+])Area under peak Mgat3 +/+ Mgat3 T37/T37 Mgat3 +/+ Mgat3 Δ/Δ %Biantennary G2Gn2M3Gn2F1810.62.95.73.03.2 G2Gn2(F)M3Gn2F1956.18.69.7 G2Gn2(F2)M3Gn2F2102.93.819.121.3*G1Gn2(Gn)M3Gn2F1851.77.47.2*G2Gn2(Gn)M3Gn2F2013.83.3*G1Gn2(F)(Gn)M3Gn2F1997.88.37.1*G2Gn2(F)(Gn)M3Gn2F21606.37.3*G2Gn2(F2)(Gn)M3Gn2F2306.125.620.8Triantennary G3Gn3(F2)M3Gn2F2468.23.65.1 G3Gn3(F3)M3Gn2F2614.48.410.7*G3Gn3(F2)(Gn)M3Gn2F2671.43.23.4*G3Gn3(F3)(Gn)M3Gn2F2817.58.39.1Tetraantennary G4Gn4(F2)M3Gn2F2833.62.92.2 G4Gn4(F3)M3Gn2F2979.73.73.3 G4Gn4(F4)M3Gn2F3125.85.75.5*G4Gn4(F2)(Gn)M3Gn2F3036.81.8*G4Gn4(F3)(Gn)M3Gn2F3182.92.1*G4Gn4(F4)(Gn)M3Gn2F3329.12.83.2Shown is a summary of MALDI-TOF spectra in Fig. 2. N-Glycan structures are written with the nonreducing end at the left.G, Gal; Gn, GlcNAc; M, Man; F, Fuc; Gn, bisecting GlcNAc; *, structures with a bisecting GlcNAc. Predicted masses are given; observed
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