In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2.

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis.

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

The Golgi Glycoprotein MGAT4D is an Intrinsic Protector of Testicular Germ Cells From Mild Heat Stress.

Male germ cells are sensitive to heat stress and testes must be maintained outside the body for optimal fertility. However, no germ cell intrinsic mechanism that protects from heat has been reported. Here, we identify the germ cell specific Golgi glycoprotein MGAT4D as a protector of male germ cells from heat stress. Mgat4d is highly expressed in spermatocytes and spermatids. Unexpectedly, when the Mgat4d gene was inactivated globally or conditionally in spermatogonia, or mis-expressed in spermatogonia, spermatocytes or spermatids, neither spermatogenesis nor fertility were affected. On the other hand, when males were subjected to mild heat stress of the testis (43 °C for 25 min), germ cells with inactivated Mgat4d were markedly more sensitive to the effects of heat stress, and transgenic mice expressing Mgat4d were partially protected from heat stress. Germ cells lacking Mgat4d generally mounted a similar heat shock response to control germ cells, but could not maintain that response. Several pathways activated by heat stress in wild type were induced to a lesser extent in Mgat4d[-/-] heat-stressed germ cells (NFκB response, TNF and TGFß signaling, Hif1α and Myc genes). Thus, the Golgi glycoprotein MGAT4D is a novel, intrinsic protector of male germ cells from heat stress.

A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice.

BACKGROUND: Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. RESULTS: We now report that after backcrossing to C57BL/6 J mice for 11-15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. CONCLUSION: We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

MGAT1 and Complex N-Glycans Regulate ERK Signaling During Spermatogenesis.

Mechanisms that regulate spermatogenesis in mice are important to define as they often apply to fertility in man. We previously showed that conditional deletion of the mouse Mgat1 gene (Mgat1 cKO) in spermatogonia causes a germ-cell autonomous defect leading to infertility. MGAT1 is the N-acetylglucosaminyltransferase (GlcNAcT-I) that initiates the synthesis of complex N-glycans. Mechanistic bases of MGAT1 loss were investigated in germ cells from 22- and 23-day males, before any changes in germ cell morphology were apparent. Gene expression changes induced by deletion of Mgat1 were determined using the Affymetrix gene chip Mouse Mogene 2.0 ST array, and relationships were investigated by bioinformatics including Gene Ontology (GO), Ingenuity Pathway Analysis (IPA), and Gene Set Enrichment Analysis (GSEA). The loss of complex N-glycans promoted the premature up-regulation of genes normally expressed later in spermatogenesis and spermiogenesis, and IPA and GSEA implicated ERK signaling. EGFR and PDGFRA transcripts and ERK1/2 signaling were reduced in 22-day Mgat1 cKO germ cells. Basigin, a germ cell target of MGAT1, activated ERK1/2 in CHO cells, but not in a Lec1 CHO mutant that lacks MGAT1 and complex N-glycans. Thus, MGAT1 is required to regulate ERK1/2 signaling during spermatogenesis, potentially via different mechanisms.

O-GlcNAc on NOTCH1 EGF repeats regulates ligand-induced Notch signaling and vascular development in mammals.

The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt-/- retina, and Notch target gene expression was decreased in Eogt-/-endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.

GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis.

Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z approximately 13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Gal beta1-4GlcNAc)(n) units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis(x) and sialyl-Lewis(x) determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.

Mutational and functional analysis of Large in a novel CHO glycosylation mutant.

Inactivating mutations of Large reduce the functional glycosylation of alpha-dystroglycan (alpha-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase beta1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, alpha-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the beta1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional alpha-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional alpha-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated alpha-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of alpha-DG.

Rectal misoprostol vs 15-methyl prostaglandin F2alpha for retained placenta after second-trimester delivery.

OBJECTIVE: The purpose of this study was to compare rectal misoprostol (RM) with 15-methyl prostaglandin F2alpha (PGF(2alpha)) for the management of retained placenta after second-trimester deliveries. STUDY DESIGN: A retrospective study of all second-trimester deliveries between the years 2000 and 2005 was performed. Women were divided into 2 groups, depending on whether they received RM or PGF(2alpha) after the delivery. RESULTS: Three hundred three second-trimester deliveries were analyzed. The time from the administration of medications to the placental delivery was significantly shorter in women who received PGF(2alpha), compared with the RM group (49.5 vs 89 minutes; P < .01). Women who received PGF(2alpha) had lower rates of retained placenta (4.9% vs 12.4%; P = .02). CONCLUSION: The use of PGF(2alpha) after second-trimester deliveries results in shorter third stage of labor and lower rates of retained placenta compared with RM.

Molecular analysis of three gain-of-function CHO mutants that add the bisecting GlcNAc to N-glycans.

LEC10 Chinese hamster ovary (CHO) cells are gain-of-function mutants that express N-acetylglucosaminyltransferase III (GlcNAc-TIII), the glycosyltransferase that adds the bisecting GlcNAc to complex N-glycans. LEC10 cells are useful for glycosylation engineering of recombinant glycoproteins, including antibody therapeutics, for defining lectin recognition specificities and for determining biological functions of the bisecting GlcNAc. We show that three CHO mutants, LEC10, LEC10A, and LEC10B, arose due to transcriptional activation of the quiescent CHO Mgat3 gene. They each express Mgat3 gene transcripts of approximately 4.7 kb at different levels (LEC10B > LEC10 > LEC10A). Southern analyses gave a single band in LEC10, LEC10A, and parent CHO DNA with four restriction enzymes but an additional band with three of them in LEC10B genomic DNA, indicative of a duplication event in LEC10B. The deduced amino acid sequence of the Mgat3 gene expressed in each CHO mutant and in parent CHO genomic DNA is identical. However, 5' UTR sequences differ with LEC10 and LEC10B containing a 5' UTR segment of the Atf4 gene downstream of the Mgat3 gene in human and mouse. Somatic cell hybrid analysis indicated that the LEC10B Mgat3 gene was induced by a cis mechanism. LEC10B glycoproteins bound more erythroagglutinin lectin (E-PHA) than LEC10 glycoproteins and MALDI-TOF MS revealed a broad spectrum of complex, bisected N-glycans expressed by the LEC10B mutant. LEC10B is therefore the cell line of choice for producing recombinant glycoproteins carrying bisected N-glycans and for investigating biological functions of the bisecting GlcNAc.

The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in alpha-glucosidase I that give rise to congenital disorder of glycosylation IIb (CDG IIb).

Lec23 Chinese hamster ovary cells are defective in alpha-glucosidase I activity, which removes the distal alpha(1,2)-linked glucose residue from Glc(3)Man(9)GlcNAc(2) moieties attached to glycoproteins in the endoplasmic reticulum. Mutations in the human GCS1 gene give rise to the congenital disorder of glycosylation termed CDG IIb. Lec23 mutant cells have been shown to alter lectin binding and to synthesize predominantly oligomannosyl N-glycans on endogenous glycoproteins. A single point mutation (TCC to TTC; Ser to Phe) was identified in Lec23 Gcs1 cDNA and genomic DNA. Serine at the analogous position is highly conserved in all GCS1 gene homologues. A human GCS1 cDNA reverted the Lec23 phenotype, whereas GCS1 cDNA carrying the lec23 mutation (S440F in human) did not. By contrast, GCS1 cDNA with an R486T or F652L CDG IIb mutation gave substantial rescue of the Lec23 phenotype. Nevertheless, in vitro assays of each enzyme gave no detectable alpha-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates alpha-glucosidase I both in vitro and in vivo. However, the S440F alpha-glucosidase I may have a small amount of alpha-glucosidase I activity in vivo based on the low levels of complex N-glycans in Lec23. A sensitive test for complex N-glycans showed the presence of polysialic acid on the neural cell adhesion molecule. The Lec23 Chinese hamster ovary mutant represents a sensitive host for detecting a wide range of mutations in human GCS1 that give rise to CDG IIb.

A mutation causing a reduced level of expression of six beta4-galactosyltransferase genes is the basis of the Lec19 CHO glycosylation mutant.

To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. Ricin(II)-agarose lectin affinity chromatography, oligomapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the beta4GalT-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an approximately 2-4-fold reduction in the level of transcripts of all six beta4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian beta4GalT genes.