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31.
G D Holt R S Haltiwanger C R Torres G W Hart 《The Journal of biological chemistry》1987,262(31):14847-14850
Previously we reported that the novel protein-saccharide linkage, O-linked N-acetylglucosamine (GlcNAc), is found in abundance on proteins associated with the cytoplasmic and nucleoplasmic faces of the nuclear pore complex. Here we demonstrate that O-GlcNAc moieties are also added to human erythrocyte cytoplasmic proteins. Intact or permeabilized erythrocytes, as well as subcellular fractions, were labeled with bovine milk galactosyltransferase and UDP-[3H] galactose. The proportion of the incorporated label found on O-GlcNAc was determined by a variety of chemical and enzymatic techniques. The bulk of the O-GlcNAc residues are found in the cytoplasm of erythrocytes, the majority of which are on an as yet unidentified 65-kDa protein. In addition, we have determined that Band 4.1, a protein which serves as a bridge joining the cytoskeleton to the inner surface of the plasma membrane in erythrocytes, also contains O-GlcNAc moieties. One of the sites of O-GlcNAc addition has been localized to the last 117 amino acids of the carboxy terminus of Band 4.1. 相似文献
32.
Hans Bakker Takuji Oka Angel Ashikov Ajit Yadav Monika Berger Nadia A. Rana Xiaomei Bai Yoshifumi Jigami Robert S. Haltiwanger Jeffrey D. Esko Rita Gerardy-Schahn 《The Journal of biological chemistry》2009,284(4):2576-2583
In mammals, xylose is found as the first sugar residue of the
tetrasaccharide
GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the
formation of the glycosaminoglycans heparin/heparan sulfate and
chondroitin/dermatan sulfate. It is also found in the trisaccharide
Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor
repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes
the formation of the UDP-xylose substrate for the different
xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in
the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place
in these organelles, no obvious requirement exists for membrane transport of
UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has
been documented, and we recently succeeded with the cloning of a human
UDP-xylose transporter (SLC25B4). Here we provide new evidence for a
functional role of UDP-xylose transport by characterization of a new Chinese
hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The
mutant fails to initiate glycosaminoglycan synthesis and is not capable of
xylosylating Notch. Complementation was achieved by expression of a
cytoplasmic variant of UXS, which proves the existence of a functional Golgi
UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid
occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated
control of the cytoplasmically localized UDP-glucose dehydrogenase in the
mutant. The data presented in this study suggest the bidirectional transport
of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in
controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First,
xylose is the starting sugar residue of the common tetrasaccharide,
GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to
proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans
(GAGs)2
(1). Second, xylose is found in
the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in
epidermal growth factor (EGF)-like repeats of proteins, such as blood
coagulation factors VII and IX
(2) and Notch
(3)
(Fig. 1). Two variants of
O-xylosyltransferases (XylT1 and XylT2) are responsible for the
initiation of glycosaminoglycan biosynthesis, which differ in terms of
acceptor specificity and tissue distribution
(4-7),
and two different enzymatic activities have been identified that catalyze
xylosylation of O-glucose residues added to EGF repeats
(8-10).
On Notch, O-glucose occurs on EGF repeats in a similar fashion as
O-fucose, which modifications have been shown to influence
ligand-mediated Notch signaling
(11-16).
Recently, rumi, the gene encoding the Notch
O-glucosyltransferase in Drosophila, has been identified,
and inactivation of the gene was found to cause a temperature-sensitive
Notch phenotype (17).
Although this finding clearly demonstrated that O-glucosylation is
essential for Notch signaling, the importance of xylosylation for Notch
functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is
synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA,
and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is
inhibited by the product of the second enzyme, UDP-Xyl
(42). B, in mammals,
UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate
for different xylosyltransferases incorporating xylose in the
glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked
glycans. The nucleotide sugar transporter SLC35D1
(52) has been shown to
transport UDP-GlcA over the ER membrane and SLC35B4
(29) to transport UDP-Xyl over
the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in
GAG biosynthesis have been isolated by screening for reduced incorporation of
sulfate (18) and reduced
binding of fibroblast growth factor 2 (FGF-2)
(19,
20) and by direct selection
with FGF-2 conjugated to the plant cytotoxin saporin
(21). Isolated cells (called
pgs, for proteoglycan synthesis mutants)
(21) exhibited defects in
various stages of GAG biosynthesis, ranging from the initiating
xylosyltransferase to specific sulfation reactions
(18,
19,
21-25).
Mutants that affect overall GAG biosynthesis were shown to have a defect in
the assembly of the common core tetrasaccharide. Interestingly, these latter
mutants could be separated into clones in which GAG biosynthesis can be
restored by the external addition of xylosides as artificial primers and those
that cannot (18). The two
mutants belonging to the first group are pgsA-745 and pgsB-761. Although
pgs-745 is defective in XylT2
(4-6,
18), pgsB-761 exhibits a
defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the
first step in the elongation of the xylosylated protein (25 (see
Fig. 1B). Restoration
of GAG biosynthesis in the latter mutant presumably occurs through a second
β1-4-galactosyltransferase, able to act on xylosides when provided at
high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the
xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase
(UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes
the synthesis of UDP-Xyl, the common donor substrate for the different
xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly,
UXS in the animal cell is localized in the lumen of the ER and/or Golgi
(26-28),
superseding at first sight the need for the Golgi UDP-xylose transporter,
which has been recently cloned and characterized
(29). Using this cell variant,
experiments were designed that establish the functional significance of
UDP-Xyl transport with respect to UDP-glucuronic acid production and
xylosylation. 相似文献
33.
Maya K. Sethi Falk F. R. Buettner Vadim B. Krylov Hideyuki Takeuchi Nikolay E. Nifantiev Robert S. Haltiwanger Rita Gerardy-Schahn Hans Bakker 《The Journal of biological chemistry》2010,285(3):1582-1586
The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments. 相似文献
34.
Robson Sartorello Alexandre Budu Piero Bagnaresi Carlos AH Fernandes Paloma M. Sato Vânia B. Bueno Marcos RM Fontes Pedro L. Oliveira Gabriela O. Paiva‐Silva Simone V. Alves Luis ES Netto Luiz H. Catalani Celia RS Garcia 《Cell biology international》2010,34(8):859-865
The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole. 相似文献
35.
Hideyuki Takeuchi Robert S. Haltiwanger 《Seminars in cell & developmental biology》2010,21(6):638-645
The Notch pathway is one of the major signaling pathways required for proper development in metazoans. Notch activity is regulated at numerous levels, and increasing evidence reveals the importance of “protein glycosylation” (modification of Notch receptors with sugars) for its regulation. In this review we summarize the significance of the Notch pathway in development and the players responsible for its glycosylation, and then discuss the molecular mechanisms by which protein glycosylation may regulate Notch function. 相似文献
36.
Hatsell S Medina L Merola J Haltiwanger R Cowin P 《The Journal of biological chemistry》2003,278(39):37745-37752
37.
P. MARTIN SANDER F.L.S. MICHAEL B. LEITE THOMAS MÖRS WINFRIED SANTEL WIGHART von KOENIGSWALD 《Zoological Journal of the Linnean Society》1994,110(2):141-179
Morphology and schmelzmuster of rootless cheek teeth of 25 extant rodent genera were studied in relation to jaw movement. A differentiation between leading and trailing edges is observed regularly in enamel thickness and schmelzmuster. Similarities between antagonists are interpreted as 'functional symmetries'. Differences in the enamel thickness, the schmelzmuster and orientation of cutting edges are controlled by functional and phylogenetic constraints. The heterogenous sample allows discrimination between these two constraints. The most obvious functional constraint leads to the almost regular occurrence of radial enamel on the push sides of cutting edges. The degree of functional symmetry seems to be determined by phylogenetic limitations. 相似文献
38.
Block of outward current in cardiac purkinje fibers by injection of quaternary ammonium ions 总被引:6,自引:5,他引:1
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We have studied the effects of iontophoretic injection of the quaternary ammonium compounds tetraethylammonium (TEA) and tetrabutylammonium (TBA) in cardiac purkinje fibers. We find that TBA(+) is a more effective blocker than TEA(+), but injection of either compound reduces the time-dependent outward plateau currents, transient outward current (I(to)), and the delayed rectifier (I(x)). Our findings provide evidence that these outward cardiac currents are carried by channels that in some respects are pharmacologically similar to squid axon potassium channels. We demonstrate that this procedure is a new tool that can be useful in the analysis of membrane currents in the heart. 相似文献
39.
IB Masters MM Eastburn PW Francis R Wootton PV Zimmerman RS Ware AB Chang 《Respiratory research》2005,6(1):16
Background
Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.Methods
Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.Results
Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.Conclusion
We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements. 相似文献40.