2Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, Glycobiology Research and Training Center, 9500 Gilman Drive-0625, University of California San Diego, La Jolla, CA 92093, USA; 3Program in Structural Biology and Biochemistry, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 4Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, England; 5Department of Pathology, University of California San Diego, La Jolla, CA 92093, USA; 6Department of Medicine, Glycobiology Research and Training Center, University of California San Diego, La Jolla, CA 92093, USA; 7Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA; 8Superfund Basic Research Center, University of California San Diego, La Jolla, CA, 92093 USA; 9Department of Paediatrics, Centre for Metabolic Disease, University of Leuven, Leuven, Belgium; 10Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, CO, USA
Received on July 24, 2001; revised on August 30, 2001; accepted on August 31, 2001.
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Abstract |
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Key words: CDG-IIa/disease/genetics/glycosylation/N-glycans
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Introduction |
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A second class of CDG (type II) has also been identified by aberrant serum transferrin mobility wherein the defect is in the processing and diversification of protein-bound N-glycan structure, and not the efficiency of N-glycosylation site usage or transit through the secretory pathway (Jaeken et al., 1994). CDG type IIa (CDG-IIa) is due to an autosomal recessive lesion in the human MGAT2 gene, residing on chromosome 14 at q21, which encodes UDP-GlcNAc:
-6-D-mannoside ß-1,2-N-acetylglucosaminyltransferase II (GlcNAcT-II, E.C.2.4.1.143), a Golgi-bound glycosyltransferase essential for the production of complex-type N-glycans present in the Golgi, on cell surfaces, and among extracellular compartments (Charuk et al., 1995
; Tan et al., 1996
; Schachter and Jaeken, 1999
). Only four patients have been described at this writing, and the oldest is now 20 years of age. In two of those cases the diagnosis was made after the age of 8 years (Jaeken et al., 1994
; Engelhardt et al., 1999
; Cormier-Daire et al., 2000
).
CDG-IIa indications include a general failure to thrive, dysmorphic facial features, feeding difficulties, and psychomotor retardation. Patients are incapable of speech and undergo occasional epileptic seizures. Also observed are osteopenia, kyphoscoliosis, blood coagulopathies, immune defects, gastrointestinal abnormalities, ventricular septal defect of the heart, and susceptibility to infection. Studies of cells from patients with CDG-IIa reveal deficiency in complex N-glycans and a predominance of hybrid-type structures that include terminal mannose linkages. Four mutations in the human MGAT2 gene have been described thus far, all of which reside in the catalytic domain of GlcNAcT-II (Ser290Phe; His262Arg; Asn318Asp; Cys339Ter). Mutations analyzed in vitro by recombinant approaches thus far have shown that each abolishes enzyme activity and may decrease protein stability (Tan et al., 1996).
Inherited genetic alterations in carbohydrate synthesis exist among mammalian species and many glycan structures differ between mice and humans. Examples include the ABO blood group antigens (Koda et al., 2000), the
1-3Gal xenotransplantation antigen (Galili and Swanson, 1991
), and the dominance of N-acetyl sialic acid in humans due to the null mutation in the N-glycolylneuraminic acid synthase gene (Chou et al., 1998
). Such genetic events cause widespread alterations of terminal saccharide linkages that are present on various glycan branches. These alterations may have substantial consequences since terminal saccharide linkages can be essential in defining the physiologic functions of glycan branches. Expression of glycan branches and terminal structures can be altered in normal and aberrant physiology by transcriptional mechanisms acting on glycosyltransferase gene promoter sequences. Moreover, glycosyltransferase expression patterns and not substrate specificity per se can be a key factor in determining physiologic function. For example, an inherited genetic mutation in the glycosyltransferase gene Galgt2 alters cell lineage expression and in this manner creates a novel function involving the homeostasis of von Willebrand factor in circulation (Mohlke et al., 1999
).
Most glycosyltransferases, including GlcNAcT-II, exhibit a high degree of specificity for glycan substrates from data obtained in in vitro enzymatic studies, and therefore may not be influenced by glycoprotein amino acid sequence contexts. Moreover, because some glycosyltransferases can compete for the same glycan substrates, the outcome involving modification of a glycoprotein in the milieu of various glycosyltransferases is at present difficult to predict (Schachter, 1991). Glycans further provide essential components of pathogen receptors in hosts, and glycosyltransferase alterations during phylogeny may reflect an evolutionary "arms race" ascribed to such pathogenhost interactions (Gagneux and Varki, 1999
). For example, the expression of
2-6 sialic acid linkages on red blood cells and epithelium differs between mice and humans; this difference can influence the tissue and species tropism of viral infections, such as influenza (Carroll et al., 1981
; Suzuki et al., 2000
).
These features of glycan expression and biosynthesis render uncertain the degree to which glycosyltransferases are subject to the axiom that evolutionary conservation of gene sequences and biochemical activity reflects conservation of physiologic function. Comparing two mammalian species with the same genetic deficiency in the N-glycan repertoire would provide an assessment of the degree of conservation of N-glycan function and may lead to increased understanding of N-glycan structurefunction relationships. In addition, studies recapitulating a genetic lesion causing CDGs may provide insights into the pathogenesis of human CDG syndromes.
We have therefore generated a complex N-glycan deficiency by inactivating the mouse Mgat2 gene, and compared phenotypes with human CDG-IIa patients. Our findings reveal that there is significant conservation of complex-type N-glycan branch function between these two species with aberrant gastrointestinal, osteogenic, and hematologic processes that promote disease. In the course of our studies, we have also developed a simpler, more accurate biochemical diagnostic test for CDG-IIa. Unexpectedly, we have discovered the presence of genetic modifiers of disease signs in the mouse genome and a novel N-glycan branch structure induced by the absence of the Mgat2 gene. These results also suggest that most human CDG-IIa cases may be undiagnosed or misdiagnosed and may represent a cause of early human infant lethality.
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Results |
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We further assessed N-glycan structures by histochemistry using the plant lectin L-PHA (Figure 2D). Organs and tissues from Mgat2-deficient mice appeared normal; however these tissues did not bind L-PHA lectin, whereas wild-type tissues did, indicating a deficiency in GlcNAcT-V N-glycan branch formation. This is consistent with the loss of complex N-glycans as GlcNAcT-II activity is essential for GlcNAcT-V substrate formation. Some remaining L-PHA binding was present in the intestine, a tissue that also retained a low level of GlcNAcT-II activity. These findings indicate that Mgat2 is required for complex N-glycan biosynthesis in vivo, but do not establish the exact structures that remain. Biophysical analyses of N-glycan structures by mass spectrometric methods were undertaken to confirm these findings and to provide insights into N-glycan biosynthesis in vivo in the absence of Mgat2 function (see Results).
Dysmorphic features and severe locomotor retardation
Those Mgat2-null mice surviving to at least 8 days of age were approximately half the size of littermates that retained a functional Mgat2 allele. They exhibited dysmorphic facial features and severe locomotor deficits. Reduced muscular development was apparent, as was a hunched spinal column, indicating scoliosis (Figure 3A). Transient paralysis and tremors similar to epileptic seizures were observed in approximately 20% of Mgat2-null mice. Further histologic examinations revealed that the brain developed normally and the various anatomical subregions, including the cortex, hippocampus, olfactory bulb, and brainstem structures, were unremarkable. Additionally, the cerebellum was of proportional size and contained a normal complement of viable Purkinje cells (data not shown).
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Thus synaptic transmission appeared to be enhanced in Mgat2-nulls versus either wild-type or heterozygous littermates. Although this was initially unexpected given the motor deficits characteristic of many of the Mgat2-null animals, we believe the explanation to be a straightforward effect of reduced muscle fiber diameter resulting from the retarded growth of null animals. As with most other anatomical structures, Mgat2-null muscle fibers were of smaller dimensions when viewed microscopically (not shown). This readily accounts for the increased amplitudes of minis and evoked responses observed, as it has been previously demonstrated that the increased longitudinal internal resistance of smaller-diameter muscle fibers in early pre- and postnatal development results in larger mini and evoked synaptic potentials recorded at the endplate (Katz and Thesleff, 1957). In addition, the reduced quantal content of evoked responses in Mgat2-nulls may be related to smaller endplate areas expected in these muscle fibers, such as occur in early development (Bennett and Florin, 1974
). Similar explanations have been given for changes in neuromuscular transmission seen in diaphragm and other skeletal muscles of growth-retarded athymic nude mice (Schofield and Marshall, 1980
).
Osteopenia with increased osteoclast activity
A significant defect in bone formation was noted in Mgat2-null mice. An observed twisting of the hunched spinal column indicated kyphoscoliosis. The entire skeletal system was affected and bones that did develop were poorly calcified and brittle, including the vertebrae, ribs, femur, hips, and skull (Figure 4A). Bone density was quantitatively determined by autoradiographic methods and was found reduced by over 30% among Mgat2-null mice (Figure 4B). Joints were also poorly articulated, and fractures were noted in some cases. The formation of secondary ossification sites was delayed such that they were absent at embryonic day 13 in the Mgat2-null mice (Figure 4C). Additionally, osteoclast-derived tartrate acid resistant phosphatase activity (TRAP) was found elevated in the femurs (Figure 4D). Normal osteoblast activity was observed (not shown), together suggesting that the net deficiency of bone density is due to increased resorption by osteoclasts.
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Factors that regulate blood coagulation are altered among human CDG-IIa patients and may predispose them to thrombosis. In Mgat2-null mice, we observed decreases in most procoagulant (clotting) factors and anticoagulant factors protein C and antithrombin, suggesting a defect in liver protein synthesis or release mechanisms (Figure 5C). However, the increased fibrinogen and plasminogen levels and the normal alkaline phosphatase and ALT activities are not consistent with severe liver dysfunction. Markedly decreased levels of protein C and antithrombin relative to the mild decreases in procoagulant factors may predispose these animals to thromboembolic diseases. Furthermore, the increased level of plasminogen with simultaneous decreased levels of its natural inhibitor, alpha-2-antiplasmin, may lead to bleeding as the result of excessive fibrinolysis. Both human and mouse mutants have increased activated partial prothromboplastin time (13%15% increase). No change in factor VIII or von Willebrand factor was observed (data not shown). Human patients also show a reduction in protein S (60% normal) and factor IX (60% normal); however, levels of these two factors were unaltered in the mouse.
Gastrointestinal abnormalities with deficient mucus production and thrombosis
On necropsy, more than half of Mgat2-deficient mice exhibited a distended stomach filled with unprocessed food or gas, suggesting an intestinal obstruction in the region of the pylorus (Figure 6A and data not shown). Obstipation was indicated as constipation was noted, and prolapse of the rectum with bleeding occurred in several cases. Blood was also found in the lumen of the stomach and intestines in several cases (Figure 6B). Histologic examination revealed normal mucosal epithelial cells; however, a reduction in mucin levels within the mucosal Brunners glands was evident (Figure 6C). Moreover, similar histochemical studies of the pyloric glands also revealed a significant decrease in mucin levels (Figure 6D). A deficiency in mucin levels within the gastrointestinal tract can result in reduced food motility and digestion, which could lead to constipation and obstipation. We also cannot rule out that ischemic intestines may have resulted from thrombosis of the mesenteric vessels with subsequent bleeding into the intestines. Hemorrhage from submicroscopic mucosal damage not detected on histologic examination may have been contributed and exacerbated by the observed blood coagulopathy.
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We next crossed Mgat2 heterozygotes into the "outbred" ICR strain and monitored the offspring produced between generations three to five. The frequency of Mgat2-null mice surviving the first week of life did not change; however, the majority of those that did survive the first week lived beyond 4 weeks of age (Figure 7A). In fact those that survived the first week of life almost always survived for many months but remained smaller than littermates that retained a functional Mgat2 gene (Figure 7B). These Mgat2-null survivors exhibited similar disease signs as described above; however, disease was more variable among individuals and the severity was reduced (Figure 7C and data not shown).
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Low fecundity and spermatogenic failure in Mgat2-null survivors
Approximately 30% of female mice lacking the Mgat2 gene produced offspring; however, they failed to nurture their pups, and the pups died within a few days unless foster mothers were provided. In contrast, male Mgat2-null mice exhibited testicular atrophy and were invariably infertile (Figure 8A and data not shown). Spermatogenesis was disrupted and no mature sperm were found in the lumen of the seminiferous tubules (Figure 8B; top panel). Spermatogonia were present, as were spermatocytes; however, a deficiency in spermatids was observed (Figure 8B; middle and bottom panels). The absence of spermatids was confirmed by histochemical analysis with Periodic acid Schiff (PAS) reagent that binds to the acrosomal structures found on developing and mature spermatids (Figure 8C; Russell et al., 1990). The observed loss of early spermatids suggests that GlcNAcT-II activity is essential for the differentiation of spermatocytes into early spermatids. Heterozygous males were unaffected with normal sperm development and abundance (not shown). Moreover, heterozygotes produced normal frequencies of functional sperm bearing the Mgat2
haplotype as judged by genotyping their offspring resulting from breeding with normal wild-type females.
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Partial FAB mass spectra from permethylated N-glycans released from control and Mgat2-null kidney are shown in Figure 9A and 9B, respectively. These data indicate that, as expected, the family of core fucosylated bi-, tri- and tetraantennary/branched bisected complex N-glycans that occur in high abundance in the normal mouse kidney (Figure 9A; Chui et al., 2001) are absent in the Mgat2-null tissue. Instead the Mgat2-null sample is characterized by a major new family of N-glycans dominated by a structure of composition Fuc3Hex5HexNAc4 (m/z 2593) that corresponds to a core fucosylated glycan with two Lewisx antennae. Other signals present in the mutant but not the normal kidney have compositions corresponding to additional or fewer Fuc and/or Hex residues (m/z 2215, 2245, 2419, 2593, 3216). Interestingly, the majority of complex-type N-glycans observed in the wild-type kidney are bisected (Figure 9A), whereas the composition Fuc3Hex5HexNAc4 is not consistent with the presence of an unsubstituted bisecting GlcNAc in addition to two Lewisx branches.
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Nano-electrospray tandem mass spectrometry (MS/MS) experiments on selected molecular ions in the spectra from the hydrofluoric acid and exo-ß-galactosidasetreated samples provided confirmation for the two branches being linked to separate mannose residues. Thus, collisional activation of the major defucosylated product at m/z 1134 [M+2Na]2+ (corresponding to the singly charged ion at m/z 2245 in the FAB spectrum shown in Figure 9C) gave a singly charged ion at m/z 1576 and a doubly charged ion at 800 consistent with loss of Hex-HexNAc-Hex from the intact molecule, indicating that the two defucosylated branches are not attached to the same mannose. Similarly, HexNAc-Hex was liberated on collisional activation of the exo-ß-galactosidase product at m/z 1836 (see Figure 9D), which can only occur if the Hex is singly substituted with HexNAc.
These findings confirm the presence of a novel N-glycan structure with a branch on the bisected GlcNAc, as represented in Figure 9F. No other branch arrangements are consistent with the linkage data, in particular the relative abundance of 2-Man and 3,4,6-Man (Figure 0E), the MS/MS data, and the overall composition. The resistance of this glycan to jackbean exo-
-mannosidase digestion, as revealed by FAB-MS experiments and by the lack of 3,4-Man in the linkage data of the digestion products (Figure 9E), is fully consistent with previous work on bisected N-glycans (Sutton-Smith et al., 2000
; Chui et al., 2001
). Furthermore, the data support the other assignments shown in Figure 9BD, including the two Lewisx branches attached at positions 2 and 4 of the 3-arm of the core (m/z 3216). N-glycan structures in the brain did not show major signals suggesting this novel N-glycan branch, although there was a minor signal at m/z 2593, which is likely to correspond to the novel bisected branch structure observed in the kidney (not shown).
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Glomerulonephritis with immunoglobulin deposition was observed on histologic examination of the kidneys of all Mgat2-null survivors more than 6 months of age (Figure 0C). The severity increased with age, and by 9 months extensive mesangial expansion was noted that frequently occluded the lumen of glomerular capillaries. Mononuclear cell infiltrates were also observed in some cases (data not shown). Blood in the urine was visible in some aging Mgat2-null survivors, and urinalysis revealed moderate to severe proteinuria in almost all Mgat2-null survivors (Figure 0
D). Increased serum titers of autoantibodies were detected, indicating an autoimmune component of disease due to GlcNAcT-II deficiency (Figure 0
E). Several Mgat2-null mice were found to have increased bacterial titers in the gut and extensive kidney tubule dilation, suggesting the possibility of pyelonephritis in some cases (data not shown).
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Discussion |
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Differences observed in disease signs between humans and mice with GlcNAcT-II deficiency may indeed reflect species-specific functions of complex-type N-glycans, especially in examples where human CDG-IIa disease signs were not observed in the mouse. However, disease signs not reported in human CDG-IIa but present in Mgat2-null mice may be prognostic for human patients with age, as all CDG-IIa patients are children as of this writing. It is also possible that humans most severely affected may die in gestation or in early postnatal life, whereas those least affected may reach maturity with a variable subset of disease signs. Although human MGAT2 mutations thus far analyzed appear to completely disable enzymatic activity in vitro (Tan et al., 1996), heterogeneity in disease signs may also result from varied and low residual activity of mutant human GlcNAcT-II not detectable by current assays. Nevertheless, the remarkable degree of conservation in disease signs between humans and mice with GlcNAcT-II deficiency can now be considered alongside results of other mutations in the mammalian germ line that block adjacent steps in N-glycan biosynthesis to further establish structurefunction relationships involving specific glycosyltransferases and N-glycan branches.
Branch-specific N-glycan formation and function in mammals
The completion of embryonic development in the absence of Mgat2 gene function contrasts with results obtained in studies of Mgat1-null embryos. Loss of Mgat1-encoded GlcNAcT-I activity disables production of hybrid N-glycans; instead, only high-mannose types are produced. The resulting absence of both hybrid and complex N-glycan formation ablates all N-glycan branching as initiated by N-acetylglucosamine linkages and invariably results in intrauterine embryo death during E9 of ontogeny (Ioffe and Stanley, 1994; Metzler et al., 1994
). This period in embryonic development represents the first trimester in human development and is a time when cell typespecific interactions and signaling processes promote significant morphogenic events. Mgat1-null embryos are defective in neural tube development, vascularization, and exhibit a randomization of cardiac loop formation resulting a high frequency of situs inversus of the early heart. No examples of human Mgat1 mutation or GlcNAcT-I deficiency have been reported. Hybrid-type N-glycans therefore appear to represent the minimal biosynthetic N-glycan structural repertoire necessary for mammalian ontogenic processes that enable, in at least most cases, the completion of embryogenesis. However, this hybrid N-glycan repertoire is inadequate for the normal development and function of various physiologic systems, resulting in CDG-IIa.
Human and mouse embryos appear able to complete embryonic development and survive past birth in the absence of Mgat2 gene function. The frequency of homozygous Mgat2-null mouse embryos at day 15 was slightly less than normal (17% in over 500 offspring analyzed) and tissues were deficient in PHA lectin binding by embryonic day 10 (data not shown). It therefore is likely that some mouse embryos lacking Mgat2 are more severely affected and die in utero. Whether we analyzed embryos or postnatal mice, we found by using lectin histology that complex N-glycans were similarly deficient from Mgat2-null tissues in all cases (data not shown).
Curiously, low levels of GlcNAcT-II activity and complex N-glycans were observed in the intestine, a finding that requires further investigation. Some of this activity may be due to the presence of GlcNAcT-III and/or GlcNAcT-IV in the in vitro assays performed. However, we cannot rule out the presence of an unknown GlcNAcT-II isozyme. Structural analyses of the products formed in the intestine are required to resolve this issue, as was accomplished in the kidney. Finally, it remains possible that the origin of this enzyme activity toward the synthetic substrate is nonmammalian, perhaps bacterial. Thus far, however, no bacterial homologues of GlcNAcT-II have been detected in the DNA databases.
N-glycan branching in the absence of Mgat2 function
Mgat2-encoded GlcNAcT-II deficiency results in an altered structural repertoire of N-glycans with a deficiency in complex subtypes. Also as expected, GlcNAcT-II deficiency results in an increase of hybrid-type N-glycan branches due to the underlying activities of both Mgat1 gene-encoded GlcNAcT-I activity and an alpha-mannosidase-III activity acting on the Man5GlcNAc2-Asn N-glycan substrate in N-glycan synthesis. Extensive glycan analyses were performed on the kidney because the initial screening suggested the presence of unusual structures. Unexpectedly, the major glycan identified in the kidney carries a Lewisx antenna at the bisecting position (Figure 9F). This structure has not been previously identified in bisected N-glycans as they have been shown to exist with an unsubstituted bisecting GlcNAc residue.
Whether this novel N-glycan branch structure exists in appreciable levels among tissues and cell types from wild-type mammals will require further study. We consider it possible that the bisecting GlcNAc in wild-type derived N-glycans is inaccessible to one or more Golgi-resident galactosyltransferases that may be preferentially expressed in the kidney. In the absence of GlcNAc addition on the 6-arm mannose residue, these N-glycans may become substrates of such galactosyltransferase activity. This could reflect a physiologically relevant mechanism that generates N-glycan branches and terminal glycan linkages in situations where GlcNAcT-II is scarce or absent. Expression levels of GlcNAcT-II may thus be relevant in some cases; in this regard it is interesting to note that the highest levels of Mgat2 RNA expression occur in tissues highly dysfunctional in the absence of GlcNAcT-II.
The bisected branch structure observed in Mgat2 deficiency could be considered a novel "pathway," or perhaps a "kinetic artifact"; however, the more relevant issue is perhaps whether this structure in some way modulates physiologic and disease processes. This possibility may be addressed, in one manner, by analyzing the disease course in mice lacking both Mgat2 and Mgat3 genes. N-glycan branching itself may be highly essential in providing a means to generate multivalent glycan ligands for various endogenous lectin molecules. Alternatively, unique saccharide linkages and termini produced on a subset of N-glycan branches may engender specific and critical functions that cannot be synthetically duplicated on other branches.
Mouse and human CDG-IIa
A tabulated comparison of mice and humans that are deficient in GlcNAcT-II activity most clearly indicates significant conservation of complex N-glycan function (Table II; Jaeken et al., 1994; Engelhardt et al., 1999
; Cormier-Daire et al., 2000
). Failure to thrive with dysmorphic facial features and poor psychomotor development are common to both species lacking GlcNAcT-II. Human patients have been found to suffer epileptic seizures, and we noted that some mutant mice exhibited tremors and intermittent paralysis. Both species have normal cerebellar development. Moreover, brain anatomy in the mouse appeared unremarkable without evidence of neuronal or glial cell loss. Deep tendon reflexes, nerve conduction velocities, and evoked potentials in human CDG-IIa are normal and mutant mice exhibited normal synaptic transmission in spontaneous and evoked neuromuscular recordings.
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Testicular atrophy is also present in both humans and mice lacking Mgat2 gene function. Whether human males with CDG-IIa are fertile is not yet known; however, male Mgat2-null mice were invariably infertile with a defect in spermatocyte differentiation leading to an absence of spermatids and sperm. We predict that human males with CDG-IIa will also be found to be infertile and similarly aspermatic, reflecting a conserved spermatocyte differentiation event that requires complex N-glycans on one or more glycoproteins for male gamete development and viability.
Further insights into complex N-glycans in normal and disease processes were obtained from studies of blood coagulation, blood chemistry, gastrointestinal function, and the immune system. Blood coagulation factor deficiencies revealed a profile very similar to human CDG II-a and suggesting a tendency towards thrombosis. These findings imply that complex N-glycan branching in the liver is crucial in the production or stability and half-life of specific coagulation factors in circulation. These alterations could be especially relevant in situations where organ dysfunction produces excessive stress on cellular membranes, such as that observed in the gastrointestinal tract.
The diagnosis of volvulus and obstipation in human CDG-IIa indicates abnormalities in gastrointestinal function. Mice lacking Mgat2 were affected with obstipation, constipation, hemorrhage, and rectal prolapse. Volvulus was not detected in the mouse but may have been present prior to necropsy. Remarkably, a deficiency of intestinal mucin among Brunners and pyloric glands was noted. The extent of mucin deficiency directly correlated with the frequency of hemorrhage and rectal prolapse (not shown). These results indicate that gastrointestinal dysfunction in the absence of complex N-glycans may be a result of mucin deficiency, leading to defects in food transport, reduced blood glucose, reduced serum protein levels, and mucosal injury with hemorrhage exacerbated by the blood coagulopathy observed. How complex N-glycans act to promote mucin production is unclear at this time. In further comparisons, blood chemistry indicated a remarkably similar, and aberrant, AST/ALT profile among mice and humans. These results are not consistent with significant liver dysfunction but suggest hemolytic disease, possible pulmonary embolisms, and muscle damage.
Some phenotypic findings in the mouse were dissimilar to human CDG-IIa or infrequent in occurrence. Heart development appeared normal in the Mgat2-null mouse in all but one case of ventricular septal defect (not shown); however, ventricular septal defect has been found in two of the four currently identified human patients. Additionally, mutant mice were anemic with an increase in reticulocytes and a decrease in platelets. However, peripheral blood indices are reported to be normal in human CDG-IIa patients. An essential role for complex N-glycans in human erythropoiesis and erythroid function is thus not indicated, in contrast to mice lacking alpha-mannosidase-II or GlcNAcT-II. Hepatomegaly also was not observed among Mgat2-null mice, although it has been reported in human CDG-IIa. Differences in human and mouse CDG-IIa also appear to involve serum Ig production. Low levels of serum Ig have been described in CDG-IIa patients and suggest defective B cell development or function. Although Ig levels vary in the ICR strain background, and thereby complicate this analysis, those Mgat2-null mice studied did not harbor significantly reduced levels among survivors more than 6 weeks of age (data not shown).
Lymphopoiesis and immune function are, however, susceptible to loss of Mgat2 gene function. A block in pre-B cell differentiation was observed in the Mgat2-null mouse bone marrow, leading to depressed B cell numbers. T cell development in the thymus was also defective, with fewer thymocytes and mature T cell precursors present. However, these developmental defects were only partial (and perhaps temporal) in scope because peripheral lymphoid compartments were colonized with mature B and T cells in these same animals. No effect was seen on myeloid development or on cell viability. Human CDG-IIa patients are, however, prone to infections; we noted a substantial increase in bacterial titers in the gastrointestinal tract of some Mgat2-null mice. Cultures of these bacteria failed to indicate the presence of abnormal strains among mice house in a specific pathogen-free vivarium; however, yeast were found on occasion.
Kidney dysfunction and autoimmune involvement were suspected in aging Mgat2-null mice following the detection of proteinuria. Recent findings of systemic lupus erythematosuslike autoimmune disease in alpha-mannosidase-II deficient mice (Chui et al., 2001) may therefore be recapitulated in Mgat2-deficient disease, considering the adjacent downstream position of GlcNAcT-II in the N-glycan biosynthetic pathway. We found increased autoantibody titers in the serum of Mgat2-null mice. However, the degree of this increase was less than that noted in alpha-mannosidase-II deficient mice. An attenuation of the autoimmune disease phenotype would not be expected simply from the positions of alpha-mannosidase-II and GlcNAcT-II in N-glycan biosynthesis; however, systemic loss of all complex N-glycans only occurs with Mgat2 deficiency, and this could moderate the autoimmune disease component. For example, lymphocyte development and immune responses appear normal in alpha-mannosidase-II deficiency because the alternate pathway to complex N-glycans remains intact (Chui et al., 2001
). Future studies of lymphocyte immune function in the absence of Mgat2 may be informative in this regard. Moreover, Mgat5-deficient mice that lack one branch found in complex N-glycans, the formation of which depends on GlcNAcT-II function, have been described as hyperresponsive to immune stimuli (Demetriou et al., 2001
). The oldest CDG-IIa patient is presently 19 years of age, and no CDG-IIa patient has been reported thus far with glomerulonephritis, kidney dysfunction, or signs of autoimmune disease.
GlcNAcT-II glycosyltransferase as a biological modifier
Altered survival frequencies and disease penetration among Mgat2-null mice suggest a modulatory role for GlcNAcT-II and complex N-glycans in physiology. The severity of the disease in the mouse was significantly reduced when the mutation was bred into a distinct genetic background (the ICR strain). Survival frequencies from breedings undertaken indicated the presence of autosomal genetic modifiers unlinked to the Mgat2 gene in meiotic recombination. The relevance of this finding to human CDG-IIa is uncertain since the survival frequency of newborn humans with MGAT2 gene defects is unknown. All CDG-IIa cases have been diagnosed only after several years of postnatal life, and some clinical heterogeneity is present among CDG-IIa patients. Heterogeneity may also result from the presence of currently unknown mutations in MGAT2 that alter but dont fully disable GlcNAcT-II activity. Nevertheless, long-term mouse survivors may reflect those children that have survived early postnatal development but failed to thrive, were subsequently diagnosed, and are currently in clinical care. Should human genetic modifiers of CDG-IIa disease exist and cause variations in disease penetrance, a proportion of the human population with CDG-IIa may well be undiagnosed at this time. In this regard, recent measurements of allele frequencies among some European populations suggest that some forms of CDG-I are more common than are clinically recognized (Matthijs et al., 2000). Additionally, diagnosis by aberrant transferrin isoelectric focusing is not always a reliable indicator (Knopf et al., 2000
), further underscoring the view that CDG prevalence in the human population is not yet established (Kornfeld, 1998
; Freeze, 1998
).
Heterogeneity in human CDGs illustrates the modulatory nature of N-glycan functions in physiology. Even among those patients with the same mutations, CDG-I phenotypes are clinically heterogeneous, and tissue-specific alterations of N-glycans in CDGs have been reported (Dupré et al., 2000; Imtiaz et al., 2000
; De Lonlay et al., 2001
). How might this heterogeneity be produced? Factors affecting efficiency of protein N-glycosylation could be involved and contribute to what has been termed microheterogeneity of protein glycosylation. Obviously, the known depressive affect of alcohol ingestion on liver protein N-glycosylation has been the basis for the use of the serum transferrin isoelectric focusing to detect CDGs. However, endogenous factors that affect the frequency of site-specific protein glycosylation and that might thereby regulate glycoprotein function in normal contexts have not been identified at this time. Alternatively, genetic polymorphisms among substrates may also be considered. For example, gene mutations that alter peptide sequences to include or exclude an N-glycosylation site could retain glycoprotein expression while modulating physiologic function. Such alterations in glycan formation or structure can affect glycoprotein half-life, aggregation properties, and lectin-ligand bindingall of which represent mechanisms capable of modulating signal transduction events in biologic processes.
The physiologic systems defective in Mgat2 null mice were not completely disabled but instead functioned less effectively and, in some cases, aberrantly. Nevertheless, disease was typically severe and lethality was common. This is consistent with the functional theme of Golgi-produced complex N-glycans as signal modulators that alter the flow of information between viable cells. Our findings further indicate that the mouse can provide an accurate model of human disease resulting from a genetic defect in protein N-glycosylation. In this regard, efforts to increase mucin production in the gastrointestinal tract, retard bone resorption, and normalize hematologic parameters may be useful approaches in treating CDG-IIa. A curative approach however may ultimately require gene replacement therapy, unlike some CDG type I syndromes that can be treated by the addition of specific monosaccharides to the diet (Niehues et al., 1998; Marquardt et al., 1999
). In CDG-IIa, there are no deficiencies of monosaccharide donors or other building blocks in glycan metabolism. With more study and with the production of other CDG models, we are optimistic that an increase in information pertinent to CDG pathogenesis will ensue and facilitate the development of additional effective therapeutic approaches.
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Materials and methods |
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Mgat2 cloning and gene targeting
A human genomic Mgat 2 probe was used to isolate two overlapping clones from a mouse 129/SvJ genomic library and to construct a targeting vector. ES cell clones bearing both systemic and conditional deletions of the Mgat2 gene were obtained as described (Marth, 1996). To acquire offspring bearing the null mutation, chimeric mice were generated by microinjection of ES cells into C57BL/6 blastocyst-stage embryos and were bred to ZP3-Cre transgenic mice maintained on the C57BL/6 background for more than 10 generations. The mutations were further bred into the C57BL/6 or ICR strains (HSD) as described in the text.
GlcNAcT-II activity
Extracts for GlcNAc-T II assays were obtained by homogenizing tissues in buffer containing 2% Triton X-100 in the presence of protease inhibitors. GlcNAc-T II activity was assayed in 0.75 mM [Man(alpha1-6)][GlcNAc(beta1-2)Man(alpha1-3)]Man(beta-1-O)octyl (GnM3-octyl) (Dr. Hans Paulsen, Hamburg, Germany), 1.0 mM UDP-[3H]GlcNAc (6700 dpm/nmole), 12.5 mM AMP, 0.15 M GlcNAc, 0.1 M MES (pH 6.5), and 20 mM MnCl2 in a total volume of 20 µl for 3060 min at 37°C. Product formation was assayed with Sep-Pak C18 reversed phase cartridges as previously described (Metzler et al., 1994). GlcNAcT- I was assayed as for GlcNAcT-II except that the substrate was 0.50 mM [Man(alpha1-6)][Man(alpha1-3)]Man(beta-1-O)octyl (M3-octyl) (Dr. Hans Paulsen, Hamburg, Germany) and the incubation contained 0.125 M GlcNAc. All assays were done at least in duplicate.
Histology
Tissues were frozen in Optimal Cutting Temperature medium (VWR Scientific) by placing embedded samples in a dry ice/isopentane bath and sectioned at 5 µm. Bone was reacted for TRAP activity (Sigma, Cat.387). Lectin histochemistry was performed as previously described with some modification (Metzler et al., 1994; Priatel et al., 2000
). Briefly, tissue sections used for L-PHA staining (E-PHA not shown) were treated in 0.03% H2O2 in methanol for 20 min, then blocked by 1% bovine serum albumin (BSA) in phosphate buffered saline for 30 min and incubated with 10 µg/ml of biotinylated L-PHA and Conacavalin A (Vector) for 4 h. Lectin binding was detected by horseradish peroxidase/Strepavidin (Vector, ABC kit) with diaminobenzidine substrate (Calbiochem). For immunofluorescence, the sectioned tissues were stained with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse antibody specific to IgA, IgM, IgG, and C3 as described elsewhere (Chui et al., 2001
). Alizarin and alcian blue staining of cartilage and bone was accomplished as described (Hogan et al., 1994
). For hematoxylin and eosin staining, the tissues were fixed, embedded, and sectioned at 7 µm as described (Chui et al., 2001
). Prior to hematoxylin and eosin staining, long bones were decalcified and fixed in Cal-EXITMII (Fisher) for 48 h. Whole blood analysis by E-PHA binding was accomplished by blotting 5 µl on nitrocellulose and allowing the blot to dry for 30 min prior to adding biotinylated E-PHA or L-PHA (Vector) at 2 µg/ml in Tris-buffered saline (TBS, pH 7.5). Following 3x washes in TBS, binding was detected with chemiluminesence or colorimetric detection reagents as above.
Mass spectrometry
N-glycans were isolated from trypsinized detergent extracts of homogenized organs by peptide:N-glycanase F (PNGase F) treatment and subjected to hydrofluoric acid defucosylation and digestion with various exoglycosidases before analysis by FAB-MS and GC-MS as previously described (Sutton-Smith et al., 2000). Nano-electrospray-MS/MS was carried out on the Q-TOF as described previously (Teng-umnuay et al., 1998
) using collision energies in the range 50100 eV.
Locomotor and muscular function
To assess locomotor gait, each paw was coated with different color ink and the mouse was allowed to walk through a 9 cm x 35 cm x 6 cm opaque tunnel placed on a sheet of paper (Barlow et al., 1996). For the rotarod test, mice were placed on an accelerating (440 rpm over 5 min) rotating drum (3-cm diameter) for three trials with a minimum of 15 min interval between trials. The mean latency to fall over the three trials was determined. In the wire hang test, mice were allowed to grasp a 1-mm-diameter wire with their front paws and then suspended 40 cm above a padded surface. The mean latency to fall (maximum 60 s) over three trials was determined. To determine grip strength, mice were suspended by the tail and lowered until the mouse grasped the loop of the mouse grip strength meter (UGO Basile, Varese, Italy). Each mouse was then gently pulled away from the loop, and the maximum grip force exerted by the mouse before loosing its grip was recorded. Five trials were run; the mean of the middle three scores was used in the analysis. Student t-tests were used to assess the differences between the genotypes.
Nociception
The hot plate and tail flick tests were preformed in a one trial test. The mean of three trials was used as the dependent measure. The latency (30-s maximum) to exhibit either a hind-foot shake or lick after placement on a 55 °C platform was measured. The latency (10 s maximum) for each mouse to move its tail from the path of a bright photo beam was also recorded. Student t-tests were used to assess the differences between the genotypes.
Synaptic transmission
Studies used a total of six null, two wild-type, and four heterozygous mice. Ages of animals at study averaged 28 days (range 2232 days), except for one null/wild-type pair studied at 77 days. Neuromuscular transmission was characterized using the phrenic nerve/diaphragm preparation as described (Hong and Chang, 1989), except that experiments were done at room temperature using physiological Ringer solution consisting of 129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose, and 10 mM Na-PIPES buffer at pH 7.3. Using digitized records, miniature endplate potentials ("minis") were automatically detected and frequency, amplitudes and kinetics analyzed using the program MiniAnalysis (Synaptosoft, Inc.). Additionally, characterization of the neurally evoked endplate potential ("evoked response") was done on two pairs of null/heterozygote littermates. µ-Conotoxin was used to eliminate muscle contraction when recording these evoked endplate responses (Hong and Chang, 1989
).
Hematology and urinalysis
Blood from the tail vein of methoxyfluorane anesthetized mice was collected into EDTA-containing polypropylene microtubes (Becton Dickinson, NJ). Analyses of red blood cells, white blood cells, and platelet cell numbers and morphology were carried out using a Cell-Dyn 3500 Automated Analyzer (Abbott Diagnostics) programmed for mouse blood parameters. Urine was collected and analyzed as previously described (Chui et al., 2001).
Serum chemistry
Blood was collected in the absence of anticoagulants by tail bleed or cardiac puncture and was allowed to clot for several hours in plastic microtubes. The serum was collected by centrifugation. Chemistry analyses were performed using a Beckman CX-7 automated chemistry analyzer with a general CV of <5%.
Coagulation
Nine parts of whole blood collected by cardiac puncture were rapidly mixed with 1 part (v/v) of buffered citrate anticoagulant (0.06 mole/L sodium citrate plus 0.04 mole/L citric acid). Platelet poor plasma (plasma) was prepared from citrated plasma by centrifugation twice at 1800 x g for 15 min at 22°C and stored at 75°C. Normal reference mouse plasma (NMP) was prepared by pooling plasma, prepared as above, from 2030 individual C57BL/6 mice of both sexes. For the one-stage clotting assay, clotting times were determined in duplicates with an ST4 semi-automated coagulation instrument (Diagnostica Stago, NJ). Except as noted below for fibrinogen assay, test samples were diluted in HN buffer (25 mM HEPES, pH 7.5, 150 mM NaCl and 1 mg/ml BSA). The prothrombin time assay was performed by incubating 30 µl of plasma at 37°C for 3 min, followed by the addition of 60 µl of thromboplastin reagent (Thromboplastin C-Plus, Baxter, FL) prewarmed to 37°C to initiate clotting. Activated partial thromboplastin time (APTT) was performed by incubating 30 µl of plasma and 30 µl of APTT reagent (Automated APTT, Organon Technika, NC) at 37°C for 5 min followed by the addition of 30 µl of prewarmed 25 mM CaCl2 to initiate clotting. For the prothrombin (factor II) activity assay, 30 µl of test plasma was diluted 1:20 in HN buffer and incubated for 3 min at 37°C with 30 µl of a 1:1 mixture of human prothrombin-depleted plasma reagent (Diagnostica Stago, Asnieres, France) and rabbit barium-adsorbed plasma. Clotting was then initiated by the addition of 60 µl of thromboplastin C-Plus. Clotting times were converted to percent NMP prothrombin concentration from a log-log standard curve prepared with dilutions between 1:5 and 1:80 in HN-buffered NMP.
Standard curves were prepared on each day of testing. Factor VII and factor X activity assays were carried out identically except that the corresponding human factordeficient plasmas were used. Factor V activity assay was carried out identically except that 30 µl of a human factor V immunodepleted plasma reagent (American Diagnostica) was used without mixing 1:1 with barium-adsorbed rabbit plasma; the samples were diluted 1:200 in HN buffer; and the standard curves were made with dilutions in HN buffer of NMP between 1:50 to 1:1000. For the Factor VIII activity assay, 30 µl of test plasma was diluted 1:20 in HN buffer and incubated for 5 min at 37°C with 30 µl of human congenital factor VIIIdeficient plasma and 30 µl of APTT reagent. Clotting was then initiated with 30 µl of 25 mM calcium chloride. The log-log standard curves were made from NMP diluted 1:5 to 1:80 in HN buffer. The factors IX, XI, and XII activity assays were performed as described for factor VIII assays, except that the corresponding human factordeficient plasmas were used.
For the fibrinogen clotting activity assay, 60 µl of test plasma, diluted 1:10 and 1:20 in Owrens Veronal Buffer were incubated for 3 min at 37°C followed by the addition of 30 µl of bovine thrombin reagent (Dade Data-Fi thrombin reagent, Baxter, FL) to activate clotting. Clotting times were converted to fibrinogen concentration from a log-log standard curve prepared with dilutions (1:5 to 1:40 in Owrens Veronal Buffer) of standard plasma containing previously calibrated fibrinogen concentrations. The von Willebrand (vWF) antigen assay was peformed in a 96-well microtiter plate precoated overnight at 4°C with 200 µl of 10 µg/ml rabbit anti-human vWF polyclonal antibody (Dako, Denmark) prepared in 50 mM Na2CO3, pH 9.6. The wells were then blocked with 25 mM Tris, pH 7.5, 150 mM NaCl (TBS) containing 3% BSA for 2 h at 37°C. After washing several times with TBS containing 1% BSA, 100 µl aliquots of test plasmas, diluted 1:100 and 1:200 in TBS/1%BSA, were incubated in the wells for 2 h at 37°C followed by washing five times with TBS containing 0.05% Tween 20. The wells were then incubated with 100 µl of horseradish peroxidaseconjugated rabbit anti-human vWF polyclonal antibodies (Dako) diluted 1:2000 in TBS/1%BSA for 1 h at 37 °C. After washing again five times with TBS-0.05% Tween 20, the assay was developed using a peroxidase substrate (Bio-Rad, CA) according to the manufacturers instruction and quantitated at 405 nm using the Versa Max microtiter plate reader (Molecular Devices, CA). A standard curve was constructed with each plate by diluting NMP 1:10 to 1:250 in TBS/1%BSA.
The protein S antigen assay was performed as described for the vWF antigen assay but using polyclonal rabbit anti-human protein S (Dako). For the protein C activity assay, 10 µl of test plasma was diluted 1:20 in TBS containing 100 mM CsCl, were incubated in a microtiter well at 37°C for 15 min with 25 µl of 2 U/ml protein C activator (PROTAC, American Diagnostica, CT) followed by the addition of 25 µl of chromogenic substrate, S-2366, to 2.5 mM (DiaPharma, OH) and absorbance at 405 nm was measured. Absorbances were converted to percent reference mouse plasma protein C from a standard curve prepared with each plate with NMP diluted 1:4 to 1:64 in TBS-CsCl.
For the plasminogen assay, 60 µl of test plasma was diluted 1:60 in 100 mM Tris, pH 8.5, containing 8.3 mM epsilon-aminocaproic acid (EACA) (Calbiochem, CA) were incubated in a microtiter well at 37°C for 5 min. followed by the addition of 20 µl of 2500 Ploug U/ml urokinase (Calbiochem, CA) and further incubation for 60 s. Next, 100 µl of chromogenic substrate, S-2403, 1.2 mM (DiaPharma, OH) were added, and absorbance at 405 nm was measured. Absorbances were converted to percent reference mouse plasma plasminogen from a log-log standard curve prepared with each plate with dilutions of NMP (1:15 to 1:240) in Tris-EACA buffer. For the alpha-2-antiplasmin assay, 50 µl of test plasma was diluted 1:40 in TBS containing 120 mM methylamine and incubated in a microtiter well at 37°C for 5 min, followed by the addition of 50 µl of 17 µg/ml plasmin (Calbiochem) and incubation for 90 sec. Next, 50 µl of chromogenic substrate, S-2403, 0.75 mM (DiaPharma), were added and residual plasmin activity was measured by absorbance at 405 nm. Absorbances were converted to percent NMP alpha-2 antiplasmin from a standard curve prepared with each plate with dilutions of NMP (1:10 to 1:160) in TBS-methylamine.
For the antithrombin activity assay, 40 µl of test plasma samples were diluted 1:40 and 1:80 in 25 mM Hepes (pH7.5), 150 mM NaCl, and 0.1% BSA (HN/BSA) and incubated in microtiter plate wells with 40 µl of factor Xa/heparin reagent consisting of 3 µg/ml XA (Enzyme Research Lab, IN) and 10 U/ml of unfractionated heparin for 3 min at 37ºC. Subsequently, 40 µl of 1.25 mg/ml chromogenic substrate S-2765 (DiaPharma, OH) was added to each well and the color was analyzed at 405 nm. Standard curves were prepared with each plate by diluting NMP 1:20 to 1:640 in HN/BSA.
Cell isolation and flow cytometry
Single cell suspension from spleen, lymph node, thymus, and bone marrow were subjected to lysis of red blood cells by ammonium chloride. Five hundred thousand cells were labeled in a final volume of 100 µl as described previously (Priatel et al., 2000). Data were acquired using FACScan and analyzed by Cell Quest (Becton Dickinson). All antibodies were obtained from Pharmingen (CA). For PHA lectin flow cytometric analyses of whole blood, 2 µg/ml of FITC-conjugated L-PHA or E-PHA (Vector) was used.
Bone density analysis
Soft tissues were removed and skeletal components were fixed in 10% formalin for 2 days. x-ray images were taken on (Hewlett Packard 43805N Faxitron Series) together with an aluminum alloy reference wedge. The radiograph was scanned on MICROTEK ScanMaker 4, and the density was determined by digital image scanning. Bone density was referenced to results obtained with the aluminum wedge.
Autoantibody titers
Tissue homogenates were coated on 96-well plates and autoreactivity was quantified using an alkaline phosphatase-congujated anti-mouse Ig light-chain monoclonal antibody and microplate reader at 405 nm as described previously (Chui et al., 2001
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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