- Email: [email protected]
Mice Deficient in Glucuronyltransferase-I
Mice Deficient in Glucuronyltransferase-I Tomomi Izumikawa and Hiroshi Kitagawa Department of Biochemistry, Kobe Pharmaceutical University, Kobe, Japan
I. II. III. IV. V. VI. VII.
Introduction ................................................................................ GlcAT-I Gene Structure.................................................................. cDNA and Protein Structure of GlcAT-I ............................................. Enzymatic Activities....................................................................... Homologous Proteins ..................................................................... Expression Pattern of GlcAT-I .......................................................... GlcAT-I in Mouse Early Embryogenesis ............................................. A. Targeted Disruption of GlcAT-I Results in Early Embryonic Lethality.......................................................... B. GlcAT-I is Essential for Embryonic Cytokinesis and Cell Division ........ C. Expression of CS and HS in Mouse Embryos .................................. D. Inactivation of GlcAT-I Results in Defective CS and HS .................... E. CS Chains are Involved in Controlling Embryonic Cell Division and Cytokinesis ............................................................................. VIII. Concluding Remarks...................................................................... References ..................................................................................
20 21 21 23 24 24 25 25 26 27 29 30 30 32
Chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (Hep) are a class of glycosaminoglycans (GAGs) that are distributed on the surface of virtually all cells and in the extracellular matrices. CS/DS and HS/Hep chains share a common carbohydrate–protein linkage region structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser. Glucuronyl transfer to the Gal residue, the final biosynthetic step in the common linkage region, is catalyzed by a key enzyme, b1,3-glucuronyltransferase, which is termed glucuronyltransferase I (GlcAT-I). As it has been reported that the expression level of GlcAT-I correlates well with the amount of GAGs, GlcAT-I is thought to regulate the expression of GAGs. In fact, a defect in the squashed vulva 8 (sqv-8) gene which encodes GlcAT-I in Caenorhabditis elegans eliminates the expression of GAGs and the mutant worms show not only a perturbation in vulval invagination but also a defect in the cytokinesis in fertilized eggs, resulting in alternating cell division and cell fusion. Here, we summarize the recent knowledge on the roles of GlcAT-I in mammalian GAG biosynthesis and embryonic cell division using GlcAT-I knock-out mice.
Progress in Molecular Biology and Translational Science, Vol. 93 DOI: 10.1016/S1877-1173(10)93002-0
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Copyright 2010, Elsevier Inc. All rights reserved. 1877-1173/10 $35.00
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IZUMIKAWA AND KITAGAWA
I. Introduction Chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (Hep) are a class of glycosaminoglycans (GAGs) that are distributed on the surface of virtually all cells and in the extracellular matrices. CS/DS and HS/Hep are covalently linked to a specific serine residue in the core protein and occur as CS/DS proteoglycans (PGs) and HS-PGs. Many of the physiological roles of CS/DS-PGs and HS-PGs are thought to be due to the CS/DS and HS side chains with the core proteins largely playing the role of a scaffold in order to make CS/DS and HS functionally available for binding to a variety of ligands. In fact, gene-targeting technology in vertebrates and invertebrates has led to elucidation of the physiological functions of HS during development and morphogenesis in addition to their regulation of signaling molecules. In contrast to the series of model organisms that are deficient in HS, we have generated a model that is lacking the CS backbone biosynthesis in Caenorhabditis elegans only.1 A study of these worms revealed that nonsulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage2,3; whereas HS is essential for embryonic morphogenesis in the later stages of development.4 These observations suggested that, in C. elegans, even though the structure of chondroitin is similar to that of HS, the function of chondroitin is different from that of HS.4 In mice, although the deficiency of an enzyme that synthesizes the HS backbones leads to neonatal lethality with not only abnormal organogenesis but also with aberrations in the signaling pathways of morphogens and growth factors,5,6 little is known about the roles of CS, mainly due to the unexpected redundancy of CS-synthesizing enzymes, thereby making the functional analysis of CS all the more difficult.1 CS/DS and HS/Hep chains are synthesized onto a common carbohydrate– protein linkage region structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser.7 The linkage region tetrasaccharide is formed by the sequential stepwise addition of monosaccharide residues by their respective specific glycosyltransferases, that is, xylosyltransferase, galactosyltransferase-I, galactosyltransferase-II, and glucuronyltransferase-I (GlcAT-I).8 The repeating disaccharide region, [(-4GlcAb1-4GlcNAca1-)n], of HS/Hep is synthesized on the linkage region by the HS copolymerase complex of EXT1 and EXT2.9,10 In contrast, the repeating disaccharide region [(-4GlcAb1-3GalNAcb1-)n] of CS/DS is formed on the linkage region by any two combinations of chondroitin synthases-1,11 2,12 -3,13 and the chondroitin polymerizing factor.14 Also, the functionally redundant multiple glycosyltransferases involved in CS/DS synthesis have been cloned.15,16 Thus, as previously mentioned, this redundancy makes it difficult to investigate the specific role of CS/DS during early embryogenesis in mammals.
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To clarify the functions of CS/DS during mammalian early embryogenesis, we focused on GlcAT-I. As GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine (Galb1-3Galb1-4Xylb1-O-Ser) and thus finalizes the formation of the common linkage region,17,18 GlcAT-I knockout mice would show complete elimination of CS/DS as well as HS/Hep. In addition, transfection of CHO cell mutants (defective in GlcAT-I) with GlcAT-I cDNA augments GAG synthesis to levels that are approximately twofold higher than those observed in wild-type cells,19 suggesting that GlcAT-I activity is rate-limiting in GAG biosynthesis. Thus, we generated GlcAT-I knockout mice and attempted to analyze in vivo functions of GlcAT-I and CS/DS at an early developmental stage.20 This chapter will discuss the significant recent advances in our understanding of the roles of GlcAT-I in mouse GAG biosynthesis and embryonic cell division. In particular, the importance of CS in embryonic cell division and cytokinesis will be presented.
II. GlcAT-I Gene Structure The human GlcAT-I gene is located on chromosome 11q12.3, and spans approximately 7 kb of human genomic DNA.21 The transcript is encoded by five exons, and all exons contain the coding sequences indicated in Fig. 1A. In addition, exon V contains the 30 -untranslated region, which includes the polyadenylation signal AATAAA. Notably, the human genome also contains a related, processed pseudogene that covers approximately 1.4 kb of the genomic sequence and shares 95.3% nucleotide identity with exons 1–5 of GlcAT-I.21 The pseudogene is localized to chromosome 3. The mouse GlcAT-I locus is located on chromosome 19-A, is organized in a very similar fashion (Fig. 1B), and spans approximately 8.5 kb of the mouse genomic sequence. In contrast to the human genome, there is no GlcAT-I pseudogene in the mouse genome. So far, orthologues have been found at least in the zebrafish, Drosophila, and C. elegans genomes,22 suggesting the existence of conserved functions in different organisms.
III. cDNA and Protein Structure of GlcAT-I GlcAT-I activity was first detected in a chick embryo cartilage extract23 and was subsequently partially purified from embryonic chick brain24 and mouse mastocytoma cells25; however, attempts to purify GlcAT-I to homogeneity have not been successful due to its low concentration and difficulty in solubilizing the enzyme. Previously, the cDNA encoding a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope on glycoproteins (GlcAT-P) was cloned.26 GlcAT-P is a b1,3-glucuronyltransferase that utilizes glycoprotein acceptor substrates having the terminal Galb1-4GlcNAc
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IZUMIKAWA AND KITAGAWA
A Human ATG
TGA ~3.2 kb //
Exon I
II
III
IV
V
6.7 kb B Mouse ATG
TGA ~4.0 kb //
Exon I
II
III
IV
V
8.5 kb 500 bp FIG. 1. Comparison of the genomic organization of human and mouse GlcAT-I. (A) Schematic illustration of the human GlcAT-I locus on chromosome 11q12.3. GlcAT-I gene consists of five exons and spans approximately 7 kb of human genomic sequence. (B) Schematic illustration of the mouse GlcAT-I locus on chromosome 19-A. GlcAT-I gene consists of five exons and spans approximately 8.5 kb of mouse genomic sequence. Its genomic organization is very similar to that of human GlcAT-I. Exon regions are denoted by boxes. Closed boxes represent the coding sequence, and open boxes denote 50 - and 30 -untranslated sequences. Translation initiation (ATG) and termination (TGA) codons are also shown. Black horizontal bars denote introns.
sequence. Mixed-substrate experiments have indicated that GlcAT-I and GlcATP are distinct enzymes, although the two enzymatic reactions are similar.27 In view of the resemblance in the reactions catalyzed by GlcAT-I and GlcAT-P, we cloned the cDNA encoding GlcAT-I from human placenta based upon information on the amino acid sequence alignment of rat GlcAT-P with the putative proteins in C. elegans and Schistosoma mansoni.17 Subsequently, based on the sequence homology to human GlcAT-I (GeneBank TM accession number AB009598), the mouse cDNA and genes were identified (GeneBank TM accession number AB019523). The human GlcAT-I cDNA had a single open reading frame of 1005-bp coding for a 335 amino acid protein showing the characteristics of a type II transmembrane protein, with one prominent hydrophobic segment of 18 amino acid residues in the N-terminal region, and a Golgi transmembrane domain.17,28 Mouse GlcAT-I cDNA also encodes a 355 amino acid, type II transmembrane protein that was 95% identical to human GlcAT-I. The overall fold of the catalytic domain (Thr-76-Val-335) of human GlcAT-I can be divided into two subdomains, the N-terminal cofactor binding domain and the C-terminal
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substrate-binding domain.29 Both human and mouse proteins contain a DDD sequence in their C-terminal, which appear to correspond to the conserved DXD motif found in most glycosyltransferases.30 One or two N-linked glycosylation sites are present in the C-terminal part of human and mouse proteins, respectively.17 The predicted size of the human or the mouse protein is approximately 37 kDa; however, both, human and mouse, recombinant protein A-tagged GlcAT-I showed two bands. Removal of the N-glycan from both the recombinant GlcAT-I proteins by enzymatic digestion resulted in the expected single band, suggesting that one potential N-linked glycosylation site of GlcAT-I is partly utilized.
IV. Enzymatic Activities GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine, Galb13Galb1-4Xylb1-O-Ser, thus finalizing the formation of the common linkage region, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser. Although GlcAT-I was first identified in 1969, the properties of GlcAT-I were not described in detail until the cDNA encoding GlcAT-I was cloned.17,28 The properties and substrate specificities of recombinant GlcAT-I, which was expressed in COS-1 cells as a soluble, protein A chimera and purified using immunoglobulin G (IgG)Sepharose, have now been determined.18,28 Recombinant human GlcAT-I utilizes only a linkage region trisaccharide derivative, such as Galb1-3Galb1-4Xyl and Galb1-3Galb1-4Xylb1-O-Ser, and little GlcA incorporation is observed with other substrates containing a terminal GalNAc or Gal residue (i.e., polymer chondroitin), longer oligosaccharide– serines derived from the linkage region, N-acetyllactosamine, lactose, asialoorosomucoid, Galb1-3GlcNAc, Galb1-3GalNAc, and, notably, Galb1-3Galb1-Obenzyl.17,18 In addition, recombinant mouse GlcAT-I utilizes a linkage region trisaccharide derivative, such as Galb1-3Galb1-4Xyl. Thus, GlcAT-I is distinct from the enzyme termed glucuronyltransferase II, which is involved in the formation of the repeating disaccharide units of CS.11–13 The above mentioned substrate specificity indicates that the minimum structural requirement for the acceptor substrate of GlcAT-I is the trisaccharide sequence, Galb1-3Galb14Xyl (Km 80.4 mM), and that the enzyme recognizes up to the third saccharide residue (Xyl) from the nonreducing end.18 This deduced trisaccharide recognition disagrees with the finding of Wei et al.,28 who claimed that Galb13Galb1-O-napthalenemethanol and Galb1-3Galb1-O-benzyl served as acceptors of recombinant hamster GlcAT-I obtained by expression cloning. X-ray crystallographic analysis revealed the crystal structure of recombinant human GlcAT-I in the presence of both the donor-substrate product UDP and the acceptor substrate analog Galb1-3Galb1-4Xyl, and helped the identification of the key residues involved in catalysis and the binding of the two Gal residues.29
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IZUMIKAWA AND KITAGAWA
The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively.31–36 Enzyme assays showed that the synthetic molecules, Galb1-3Galb1-4Xyl(2-O-phosphate)-O-Ser and Galb1-3Gal(6-Osulfate)b1-4Xyl(2-O-phosphate)-O-Ser, were better substrates for the truncated form of the recombinant human GlcAT-I than the unmodified trisaccharide serine, whereas Gal(6-O-sulfate)b1-3Galb1-4Xyl(2-O-phosphate)-O-Ser exhibited no acceptor activity.37 The crystal structure of the catalytic domain of GlcAT-I with UDP and bound Galb1-3Gal(6-O-sulfate)b 1-4Xyl(2-O-phosphate)-O-Ser revealed that the Xyl(2-O-phosphate)-O-Ser is disordered and that the 6-O-sulfate interacts with Gln-318 from the second GlcAT-I monomer in the dimeric enzyme.29,37 The above results indicate the possible involvement of such modifications in the processing and maturation of the growing linkage-region-oligosaccharide required for the assembly of GAG chains. In addition, the sulfotransferase responsible for the 6-O-sulfation of Gal residues and the kinase responsible for the 2-O-phosphorylation of the Xyl residue in the linkage region have been identified.38,39
V. Homologous Proteins GlcAT-I belongs to a group of Golgi-associated b1,3-glucuronyltransferases whose founding member is the human HNK-1 glucuronyltransferase.26,40,41 Phylogenetic analysis showed that GlcAT-I is closely related to the homologous proteins GlcAT-P and GlcAT-S. The amino acid sequence of GlcAT-I has a proline-rich domain (from Pro-30 to Pro-75) next to the transmembrane region, as is seen in several other glycosyltransferases, including GlcAT-P and GlcAT-S, which synthesizes the precursor structure GlcAb1-3Galb1-4GlcNAc-R for the HNK-1 carbohydrate epitope GlcA(3-O-sulfate)b1-3Galb1-4GlcNAc-R.26,40,41 Database searches indicate that the amino acid sequence of GlcAT-I displays 43% and 46% sequence identity to GlcAT-P and GlcAT-S, respectively. The highest sequence identity is found in the C-terminal catalytic domain, which follows the proline-rich region (252 amino acids between Pro-68 and Glu-319 overlap with about 60% identity) 17,28 and contains the four previously identified highly conserved motifs (I–IV) of putative glucuronyltransferases among animal species.26,39
VI. Expression Pattern of GlcAT-I Northern blot analysis for GlcAT-1 mRNA demonstrated a single band of 1.5 kb in all human tissues examined.21 The GlcAT-I gene exhibited ubiquitous but differential expression in the human tissues examined. Notably,
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
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expression was particularly abundant in the placenta, followed by the pancreas, brain, and heart. In addition, Northern blot analysis of mouse mRNA demonstrated a single band of 1.8 kb (Fig. 2). The mouse GlcAT-I gene was expressed from embryonic day 7 (Fig. 2B). In adult tissues, its expression was detected in virtually all the tissues examined, with the highest levels found in the liver, brain, and kidney (Fig. 2A). These expression patterns of mouse GlcAT-I have also been previously described.28 Additionally, to establish the expression pattern of GlcAT-I during mouse embryogenesis, in situ hybridization was carried out. At embryonic day 8, GlcAT-I was expressed in the telencephalon, hindbrain, and in the frontonasal prominence. At embryonic day 16, GlcAT-I was prominently expressed in the teeth, thymus, kidney, and cartilage primordia (unpublished results). These results indicate a highly specific temporal and spatial expression pattern of GlcAT-I during embryogenesis.
VII. GlcAT-I in Mouse Early Embryogenesis A. Targeted Disruption of GlcAT-I Results in Early Embryonic Lethality
2.4
7
5
E1
kb
E1
1
B
E7 E1
kb 4.4
H
A
ea r Br t ai Sp n le Lu en ng Li ve Sk r el Ki eta dn l m e us Te y cl e st is
To examine the functions of GlcAT-I in mammalian early embryogenesis, the GlcAT-I gene was inactivated via homologous recombination in mouse ES cells.20 The targeting vector was constructed by inserting a neomycin resistance cassette into exon II (Fig. 3).
4.4 2.4
1.35 1.35 FIG. 2. Northern blot analysis of mouse GlcAT-I. Northern blots with mRNAs from various mouse tissues (A) or embryonic stages (B) were hybridized with a probe for mouse GlcAT-I cDNA. (A) Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. (B) Lane 1, embryonic day 7; lane 2, embryonic day 11; lane 3, embryonic day 15; lane 4, embryonic day 17.
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IZUMIKAWA AND KITAGAWA
SmaI
SmaI
6.4 kb
//
// Exon I 7.0 kb
EcoRI
Exon I //
II
III
Exon I
V
Wild-type allele
EcoRI
II NEO
SmaI
//
IV
EcoRI
Targeting vector
DT-A SmaI
8.0 kb II NEO
III
IV //
Targeted allele
1 kb EcoRI
8.6 kb
EcoRI
FIG. 3. Targeted disruption of mouse GlcAT-I gene.20 The neomycin resistance cassette was inserted into exon II of GlcAT-I gene. Coding and noncoding exons of GlcAT-I gene are shown by closed and open boxes, respectively, and the PGKneobpA cassette (NEO) and diphtheria toxin A fragment gene cassette (DT-A) are represented by gray boxes.
GlcAT-Iþ/– mice had an apparently normal phenotype and were born at a largely Mendelian frequency. They were intercrossed and more than 300 offspring were genotyped by PCR. No GlcAT-I–/– neonates and embryos after E6.5 could be detected, indicating that the mutant embryos died during early embryogenesis (Table I). In fact, even at E2.5 (8-cell stage), only 2% of the GlcAT-I/ embryos were detected, suggesting that most GlcAT-I/ mutants died before E2.5 (8-cell stage).20
B. GlcAT-I is Essential for Embryonic Cytokinesis and Cell Division To further analyze the lethalilty and phenotypes of the GlcAT-I–/– embryos during early development, 2-cell stage embryos, derived from heterozygous intercrosses (GlcAT-Iþ/ GlcAT-Iþ/) or other matings (GlcAT-Iþ/ GlcATIþ/þ or GlcAT-Iþ/þ GlcAT-Iþ/þ), were cultured until the blastocyst stage (Table II). The results of the in vitro culture experiments showed that 13% of the embryos from the GlcAT-Iþ/ heterozygous intercrosses died between the 2-cell and 8-cell stage, although all embryos from GlcAT-Iþ/ GlcAT-Iþ/þ or GlcAT-Iþ/þ GlcAT-Iþ/þ were viable (Table II). Notably, of the embryos from
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
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TABLE I GENOTYPE ANALYSIS OF PROGENY FROM GLCAT-I HETEROZYGOUS INTERCROSSES20 No. of progeny with genotypea Day
þ/þ
þ/–
–/–
No. resorbedb
No. total
Neonate
46 (38%)
74 (62%)
0 (0%)
–
120
E8.5
22 (39%)
31 (56%)
0 (0%)
3 (5%)
56
E7.5
12 (19%)
43 (67%)
0 (0%)
9 (14%)
64
E6.5
17 (39%)
18 (41%)
0 (0%)
9 (20%)
44
E2.5
19 (45%)
22 (53%)
1 (2%)
–
42
Percentages of different genotypes appear in parentheses. a Genotyping of each developmental stage was performed by PCR. b Resorbed embryos were not genotyped.
the heterozygous intercrosses, only 7% GlcAT-I/ embryos could be identified at the implantation stage, while the fraction of the GlcAT-Iþ/þ and the GlcATIþ/ embryos that were viable was within Mendelian expectations (1:2), confirming that GlcAT-I inactivation is lethal before the 8-cell stage. Moreover, reversion of cell division was observed in embryos only from the GlcAT-Iþ/ heterozygous intercrosses. Figure 4 shows a representative example of the reversion of cell division in one embryo from the GlcAT-Iþ/ heterozygous intercrosses. The 2-cell (E1.5) embryo divided into a 4-cell embryo, and then insufficient cytoplasmic division seemed to force the embryonic cell compartments to revert to an unusual 3-cell embryo with four nuclei. This unusual embryo eventually died, most likely due to incomplete cytokinesis (Fig. 4A and B). These results indicate that GlcAT-I function is essential for embryonic cytokinesis and cell division.
C. Expression of CS and HS in Mouse Embryos As mentioned earlier, because GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine, Galb1-3Galb1-4Xylb1-O-Ser, thus finalizing the formation of the common GAG–protein linkage region GlcAb1-3Galb1-3Galb14Xylb1-O-Ser,17 it was expected that inactivation of GlcAT-I would abolish both CS and HS chains in mouse embryos. Immunocytochemistry with wild-type mouse 2-cell embryos and blastocysts was first performed using an anti-CS (LY111) or anti-HS (Hepss-1) monoclonal antibodies. As expected, fluorescent signals were detected in all 2-cell embryos (Fig. 5C and D) and blastocysts with
TABLE II GENOTYPE ANALYSIS OF EMBRYOS CULTURED IN VITRO FROM 2-CELL-STAGE EMBRYOS TO BLASTOCYST IMPLANTATION STAGES20 No. of progeny with genotypea
No. of dead embryosb
8-cell to morula
Morula to blastocyst
Blastocyst to hatched blastocyst
Total dead embryos
No. total
Parental genotype
þ/þ
þ/–
–/–
2-cell to 8-cell
GlcAT-Iþ/ GlcAT-Iþ/
32 (29%)
55 (49%)
8 (7%)
15
0
0
2
17 (15%)
112
GlcAT-Iþ/ GlcAT-Iþ/þ
23 (59%)
14 (36%)
–
0
0
0
2
2 (5%)
39
GlcAT-Iþ/þ GlcAT-Iþ/þ
42 (98%)
–
–
0
1
0
0
1 (2 %)
43
Percentages of different genotypes appear in parentheses. a Genotyping was performed by PCR. b Dead embryos were not genotyped, but their lethal stages were determined.
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
A
E1.5
B
E2.0
E2.5
29
E3.0
Hoechst
FIG. 4. Reversion of cytokinesis in embryos from GlcAT-I heterozygous intercrosses.20 (A) E1.5 embryos were isolated from heterozygous crosses and cultured. Representative features are depicted. Reversal of cytokinesis was observed in one embryo from GlcAT-I heterozygous intercrosses (arrowhead). No reversal of cytokinesis was detected in embryos from crosses of wild-type and heterozygous mice. (B) The two embryos shown in panel (A) were stained with Hoechst 33342. The abnormal embryo (arrowheads in panel (A)) failed to complete cytokinesis and a double nucleated cell appeared (upper), whereas cell division of the other embryo continued normally (lower).
either of these antibodies; and the corresponding signals were eliminated by chondroitinase ABC (CSase) or heparitinases (HSase), respectively, indicating that both CS and HS were produced in mouse 2-cell embryos and blastocysts.20
D. Inactivation of GlcAT-I Results in Defective CS and HS Next, double immunostaining of GlcAT-I/ and GlcAT-Iþ/ embryos as well as GlcAT-Iþ/þ embryos using anti-CS (LY111) and anti-DHS (3G10) monoclonal antibodies was carried out. GlcAT-Iþ/þ and GlcAT-Iþ/ embryos were stained by both anti-CS and anti-DHS monoclonal antibodies, whereas the GlcAT-I/ embryos were not stained.20 These findings suggest that GlcATI/ embryos seem to lack both the CS and HS chains.
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IZUMIKAWA AND KITAGAWA
A
C
LY111
B
D
Hepss-1
FIG. 5. Immunocytohistochemistry of mouse 2-cell embryos using an anti-CS or anti-HS monoclonal antibody.20 Wild-type embryos at E1.5 were collected and stained. Left panels (A, B) show embryos examined by phase-contrast microscopy. Immunofluorescent staining by anti-CS (LY111) or anti-HS (Hepss-1) antibody is shown in right panels (C, D).
E. CS Chains are Involved in Controlling Embryonic Cell Division and Cytokinesis As described above, GlcAT-I/ embryos showed loss of synthesis of both CS and HS and died before the 8-cell stage due to failure of cytokinesis. However, it is not clear whether the embryonic cell death observed in the GlcAT-I/ embryos is due to deficiency of CS, HS, or both. If CS or HS is indispensable for proper embryonic cytokinesis and cell division, digestion of CS or HS at the embryonic cell surface might also induce abnormal cell division. The treatment of 2-cell embryos with CSase showed that 67% of treated embryos died between the 2-cell and 8-cell stages. In contrast, most embryos treated with heat-inactivated CSase/HSase and 65% of embryos treated with HSase developed normally in to blastocysts (Table III, Fig. 6). These results indicate that CS chains, but not HS chains, are involved in controlling embryonic cell division and cytokinesis.
VIII. Concluding Remarks It has been revealed that in C. elegans, nonsulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, while HS is essential for embryonic morphogenesis in the later stages of development.2–4 In addition, prior studies on EXT1 or EXT2 in mice demonstrated that
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TABLE III ANALYSIS OF LETHAL STAGES OF IN VITRO CULTURED EMBRYOS AFTER TREATMENT WITH CSASE OR HSASE20 No. of dead embryos
Controla
2-cell to 8-cell
8-cell to morula
Morula to blastocyst
No. of viable embryos to blastocyst stage
No. total
0
0
2
29
31
CSase treatment
22
5
0
6
33
HSase treatment
2
5
1
15
23
a
Embryos were treated with heat-inactivated CSase/HSase.
E1.5
E2.5
E2.75
E3.5
E4.0
Control
A
CSase
B
HSase
C
FIG. 6. Depletion of CS results in cytokinesis defects.20 Wild-type embryos at E1.5 were collected and incubated with heat-inactivated CSase/HSase (A, control), CSase (B), or HSase (C), respectively. Treatment of embryos with CSase showed that these two embryos died from 2-cell to 8-cell stages (B), although control (A) and HSase-treated (C) embryos developed normally to blastocysts. Representative features are depicted.
these genes are essential for HS synthesis and early development.5,6 Notably, EXT1- or EXT2-null embryos developed normally until around E6.5, when they became growth-arrested and failed to gastrulate. In addition, the marked reduction of HS in ES cells from EXT1- or EXT2-deficient mice has been reported.5,6 To clarify the roles of CS in early embryogenesis in mammals, GlcAT-I knockout mice were generated by gene targeting.20 Mice with a deletion of GlcAT-I showed marked reduction of the synthesis of CS and HS and embryonic lethality before the 8-cell stage due to failed cytokinesis.
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In addition, treatment of wild-type 2-cell embryos with CSase had marked effects on cell division, although many HSase-treated embryos normally developed to blastocysts. These results suggest that CS in mammals, as with nonsulfated chondroitin in C. elegans, is indispensable for embryonic cell division. So far, there are no reports on the role of CS or CSPG in cytokinesis and cell division in mice. To gain more insight into the role of CSPG in mammalian cell division, future studies on the identification of core proteins modified with CS, that are involved in mouse embryonic cell division, are needed. Moreover, although the GlcAT-I gene is ubiquitously expressed in virtually every human and mouse tissue examined, it exhibits marked differential expression among tissues.17,28 Thus, conditional knockout of the gene in mice would provide essential information regarding the biological function of sulfated GAGs in each tissue.
References 1. Uyama T, Kitagawa H, Sugahara K. Biosynthesis of glycosaminoglycans and proteoglycans. In: Kamerling J, editor. Comprehensive glycoscience, vol. 3. Amsterdam: Elsevier; 2007. p. 79–104. 2. Mizuguchi S, Uyama T, Kitagawa H, Nomura K, Dejima K, Gengyo-Ando K, Mitani S, Sugahara K, Nomura K. Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans. Nature 2003;423:443–8. 3. Izumikawa T, Kitagawa H, Mizuguchi S, Nomura KH, Nomura K, Tamura J, Gengyo-Angyo K, Mitani K, Sugahara K. Nematode chondroitin polymerizing factor showing cell- /organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division. J Biol Chem 2004;279:53755–61. 4. Kitagawa H, Izumikawa T, Mizuguchi S, Dejima K, Nomura KH, Egusa N, Taniguchi F, Tamura K, Gengyo-Ando K, Mitani S, Nomura K, Sugahara K. Expression of rib-1, a Caenorhabditis elegans homolog of the human tumor suppressor EXT genes, is indispensable for heparan sulfate synthesis and embryonic morphogenesis. J Biol Chem 2007;282:8533–44. 5. Lin X, Wei G, Shi Z, Dryer L, Esko JD, Wells DE, Matzuk MM. Disruption of gastrulation and heparan sulfate biosynthesis in EXT1-deficient mice. Dev Biol 2000;224:299–311. 6. Stickens D, Zak BM, Rougier N, Esko JD, Werb Z. Mice deficient in Ext2 lack heparan sulfate and develop exostoses. Development 2005;132:5055–68. 7. Lindahl U, Rode´n L. In: Gottschalk A, editor. Glycoprotein. Amsterdam: Elsevier; 1972. p. 491–517. 8. Sugahara K, Kitagawa H. Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr Opin Struct Biol 2000;10:518–27. 9. McCormick C, Leduc Y, Martindale D, Mattison K, Esford LE, Dyer AP, Tufaro F. The putative tumor suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat Genetics 1998;19:158–61. 10. Lind T, Tufaro F, McCormick C, Lindahl U, Lidholt K. The putative tumor suppressor EXT1 alters the expression of cell-surface heparan sulfate. J Biol Chem 1998;273:26265–8. 11. Kitagawa H, Uyama T, Sugahara K. Molecular cloning and expression of a human chondroitin synthase. J Biol Chem 2001;276:38721–6. 12. Izumikawa T, Uyama T, Okuura Y, Sugahara K, Kitagawa H. Involvement of chondroitin sulfate synthase-3 (chondroitin synthase-2) in chondroitin polymerization through its interaction with chondroitin synthase-1 or chondroitin-polymerizing factor. Biochem J 2007;403:545–52.
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13. Izumikawa T, Koike T, Shiozawa S, Sugahara K, Tamura J, Kitagawa H. Identification of chondroitin sulfate glucuronyltransferase as chondroitin synthase-3 involved in chondroitin polymerization: chondroitin polymerization is achieved by multiple enzyme complexes consisting of chondroitin synthase family members. J Biol Chem 2008;283:11396–406. 14. Kitagawa H, Izumikawa T, Uyama T, Sugahara K. Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization. J Biol Chem 2003;278:23666–71. 15. Uyama T, Kitagawa H, Tamura J, Sugahara K. Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J Biol Chem 2002;277:8841–6. 16. Uyama T, Kitagawa H, Tanaka J, Tamura J, Ogawa T, Sugahara K. Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate. J Biol Chem 2003;278:3072–8. 17. Kitagawa H, Tone Y, Tamura J, Neumann KW, Ogawa T, Oka S, Kawasaki T, Sugahara K. Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J Biol Chem 1998;273:6615–8. 18. Tone Y, Kitagawa H, Oka S, Kawasaki T, Sugahara K. Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. FEBS Lett 1999;459:415–20. 19. Bei X, Wei G, Sinha A, Esko JD. Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I. J Biol Chem 1999;274:13017–24. 20. Izumikawa T, Kanagawa N, Watamoto Y, Okada M, Saeki M, Sakano M, Sugahara K, Sugihara M, Asano M, Kitagawa H. Impairment of embryonic cell division and glycosaminoglycan biosynthesis in glucuronyltransferase-I-deficient mice. J Biol Chem 2010;285:12190–6. 21. Kitagawa H, Taoka M, Tone Y, Sugahara K. Human glycosaminoglycan glucuronyltransferase I gene and a related processed pseudogene: genomic structure, chromosomal mapping and characterization. Biochem J 2001;358:539–46. 22. Bulik DA, Wei G, Toyoda H, Kinoshita-Toyoda A, Waldrip WR, Esko JD, Robbins PW, Selleck SB. sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis. Proc Natl Acad Sci USA 2000;97:10838–43. 23. Helting T, Rode´n L. Biosynthesis of chondroitin sulfate II. Glucuronosyl transfer in the formation of the carbohydrate–protein linkage region. J Biol Chem 1969;244:2799–805. 24. Brandt AE, Distler J, Jourdian GW. Biosynthesis of the chondroitin sulfate-protein linkage region: purification and properties of a glucuronosyltransferase from embryonic chick brain. Proc Natl Acad Sci USA 1969;64:374–80. 25. Helting T. Biosynthesis of heparin: solubilization and partial purification of uridine diphosphate glucuronic acid: acceptor glucuronosyltransferase from mouse mastocytoma. J Biol Chem 1972;247:4327–32. 26. Terayama K, Oka S, Seiki T, Miki Y, Nakamura A, Kozutsumi Y, Takio K, Kawasaki T. Cloning and functional expression of a novel glucuronyltransferase involved in the biosynthesis of the carbohydrate epitope HNK-1. Proc Natl Acad Sci USA 1997;94:6093–8. 27. Curenton T, Ekbrog G, Rode´n L. Glucuronosyl transfer to galactose residues in the biosynthesis of HNK-1 antigens and xylose-containing glycosaminoglycans: one or two transferases? Biochem Biophys Res Commun 1991;179:416–22. 28. Wei G, Bai X, Sarkat AK, Esko JD. Formation of HNK-1 determinants and the glycosaminoglycan tetrasaccharide linkage region by UDP-GlcUA:Galactose b1,3-glucuronosyltransferases. J Biol Chem 1999;274:7857–64.
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29. Pedersen LC, Tsuchida K, Kitagawa H, Sugahara K, Darden TA, Negishi M. Heparan/chondroitin sulfate biosynthesis: structure and mechanism of human glucuronyltransferase I. J Biol Chem 2000;275:34580–5. 30. Wiggins S, Munro S. Activity of the yeast MNN1 alpha-1, 3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc Natl Acad Sci USA 1998;95:7945–50. 31. Sugahara K, Yamashina I, De Waard P, Van Halbeek H, Vliegenthart JFG. Structural studies on sulfated glycopeptides from the carbohydrate–protein linkage region of chondroitin 4-sulfate proteoglycans of swarm rat chondrosarcoma. Demonstration of the structure Gal(4-O-sulfate) beta 1-3Gal beta 1-4XYL beta 1-O-Ser. J Biol Chem 1988;263:10168–74. 32. Sugahara K, Ohi Y, Harada T, de Waard P, Vliegenthart JF. Structural studies on sulfated oligosaccharides derived from the carbohydrate–protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residues. J Biol Chem 1992;267:6027–35. 33. de Waard P, Vliegenthart JF, Harada T, Sugahara K. Structural studies on sulfated oligosaccharides derived from the carbohydrate–protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues. J Biol Chem 1992;267:6036–44. 34. Sugahara K, Ohkita Y, Shibata Y, Yoshida K, Ikegami A. Structural studies on the hexasaccharide alditols isolated from the carbohydrate–protein linkage region of dermatan sulfate proteoglycans of bovine aorta: demonstration of iduronic acid-containing components. J Biol Chem 1995;270:7204–12. 35. Lauder RM, Huckerby TN, Nieduszynski IA. Increased incidence of unsulphated and 4-sulphated residues in the chondroitin sulphate linkage region observed by high-pH anionexchange chromatography. Biochem J 2000;347:339–48. 36. De Beer T, Inui A, Tsuda H, Sugahara K, Vliegenthart JF. Polydispersity in sulfation profile of oligosaccharide alditols isolated from the protein-linkage region and the repeating disaccharide region of chondroitin 4-sulfate of bovine nasal septal cartilage. Eur J Biochem 1996;240:789–97. 37. Tone Y, Pedersen LC, Yamamoto T, Izumikawa T, Kitagawa H, Nishihara J, Tamura J, Negishi K, Sugahara K. 2-O-phosphorylation of xylose and 6-O-sulfation of galactose in the protein linkage region of glycosaminoglycans influence the glucuronyltransferase-I activity involved in the linkage region synthesis. J Biol Chem 2008;283:16801–7. 38. Kitagawa H, Tsutsumi K, Ikegami-Kuzuhara A, Nadanaka S, Goto F, Ogawa T, Sugahara K. Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1. J Biol Chem 2008;283:27438–43. 39. Koike T, Izumikawa T, Tamura J, Kitagawa H. FAM20B is a kinase that phosphorylates xylose in the glycosaminoglycan-protein linkage region. Biochem J 2009;421:157–62. 40. Seiki T, Oka S, Terayama K, Imiya K, Kawasaki T. Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope. Biochem Biophys Res Commun 1999;255:182–7. 41. Shimoda Y, Tajima Y, Nagase T, Harii K, Osumi N, Sanai Y. Cloning and expression of a novel galactoside b1,3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope. J Biol Chem 1999;274:17115–22.
I. II. III. IV. V. VI. VII.
Introduction ................................................................................ GlcAT-I Gene Structure.................................................................. cDNA and Protein Structure of GlcAT-I ............................................. Enzymatic Activities....................................................................... Homologous Proteins ..................................................................... Expression Pattern of GlcAT-I .......................................................... GlcAT-I in Mouse Early Embryogenesis ............................................. A. Targeted Disruption of GlcAT-I Results in Early Embryonic Lethality.......................................................... B. GlcAT-I is Essential for Embryonic Cytokinesis and Cell Division ........ C. Expression of CS and HS in Mouse Embryos .................................. D. Inactivation of GlcAT-I Results in Defective CS and HS .................... E. CS Chains are Involved in Controlling Embryonic Cell Division and Cytokinesis ............................................................................. VIII. Concluding Remarks...................................................................... References ..................................................................................
20 21 21 23 24 24 25 25 26 27 29 30 30 32
Chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (Hep) are a class of glycosaminoglycans (GAGs) that are distributed on the surface of virtually all cells and in the extracellular matrices. CS/DS and HS/Hep chains share a common carbohydrate–protein linkage region structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser. Glucuronyl transfer to the Gal residue, the final biosynthetic step in the common linkage region, is catalyzed by a key enzyme, b1,3-glucuronyltransferase, which is termed glucuronyltransferase I (GlcAT-I). As it has been reported that the expression level of GlcAT-I correlates well with the amount of GAGs, GlcAT-I is thought to regulate the expression of GAGs. In fact, a defect in the squashed vulva 8 (sqv-8) gene which encodes GlcAT-I in Caenorhabditis elegans eliminates the expression of GAGs and the mutant worms show not only a perturbation in vulval invagination but also a defect in the cytokinesis in fertilized eggs, resulting in alternating cell division and cell fusion. Here, we summarize the recent knowledge on the roles of GlcAT-I in mammalian GAG biosynthesis and embryonic cell division using GlcAT-I knock-out mice.
Progress in Molecular Biology and Translational Science, Vol. 93 DOI: 10.1016/S1877-1173(10)93002-0
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Copyright 2010, Elsevier Inc. All rights reserved. 1877-1173/10 $35.00
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I. Introduction Chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (Hep) are a class of glycosaminoglycans (GAGs) that are distributed on the surface of virtually all cells and in the extracellular matrices. CS/DS and HS/Hep are covalently linked to a specific serine residue in the core protein and occur as CS/DS proteoglycans (PGs) and HS-PGs. Many of the physiological roles of CS/DS-PGs and HS-PGs are thought to be due to the CS/DS and HS side chains with the core proteins largely playing the role of a scaffold in order to make CS/DS and HS functionally available for binding to a variety of ligands. In fact, gene-targeting technology in vertebrates and invertebrates has led to elucidation of the physiological functions of HS during development and morphogenesis in addition to their regulation of signaling molecules. In contrast to the series of model organisms that are deficient in HS, we have generated a model that is lacking the CS backbone biosynthesis in Caenorhabditis elegans only.1 A study of these worms revealed that nonsulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage2,3; whereas HS is essential for embryonic morphogenesis in the later stages of development.4 These observations suggested that, in C. elegans, even though the structure of chondroitin is similar to that of HS, the function of chondroitin is different from that of HS.4 In mice, although the deficiency of an enzyme that synthesizes the HS backbones leads to neonatal lethality with not only abnormal organogenesis but also with aberrations in the signaling pathways of morphogens and growth factors,5,6 little is known about the roles of CS, mainly due to the unexpected redundancy of CS-synthesizing enzymes, thereby making the functional analysis of CS all the more difficult.1 CS/DS and HS/Hep chains are synthesized onto a common carbohydrate– protein linkage region structure, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser.7 The linkage region tetrasaccharide is formed by the sequential stepwise addition of monosaccharide residues by their respective specific glycosyltransferases, that is, xylosyltransferase, galactosyltransferase-I, galactosyltransferase-II, and glucuronyltransferase-I (GlcAT-I).8 The repeating disaccharide region, [(-4GlcAb1-4GlcNAca1-)n], of HS/Hep is synthesized on the linkage region by the HS copolymerase complex of EXT1 and EXT2.9,10 In contrast, the repeating disaccharide region [(-4GlcAb1-3GalNAcb1-)n] of CS/DS is formed on the linkage region by any two combinations of chondroitin synthases-1,11 2,12 -3,13 and the chondroitin polymerizing factor.14 Also, the functionally redundant multiple glycosyltransferases involved in CS/DS synthesis have been cloned.15,16 Thus, as previously mentioned, this redundancy makes it difficult to investigate the specific role of CS/DS during early embryogenesis in mammals.
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To clarify the functions of CS/DS during mammalian early embryogenesis, we focused on GlcAT-I. As GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine (Galb1-3Galb1-4Xylb1-O-Ser) and thus finalizes the formation of the common linkage region,17,18 GlcAT-I knockout mice would show complete elimination of CS/DS as well as HS/Hep. In addition, transfection of CHO cell mutants (defective in GlcAT-I) with GlcAT-I cDNA augments GAG synthesis to levels that are approximately twofold higher than those observed in wild-type cells,19 suggesting that GlcAT-I activity is rate-limiting in GAG biosynthesis. Thus, we generated GlcAT-I knockout mice and attempted to analyze in vivo functions of GlcAT-I and CS/DS at an early developmental stage.20 This chapter will discuss the significant recent advances in our understanding of the roles of GlcAT-I in mouse GAG biosynthesis and embryonic cell division. In particular, the importance of CS in embryonic cell division and cytokinesis will be presented.
II. GlcAT-I Gene Structure The human GlcAT-I gene is located on chromosome 11q12.3, and spans approximately 7 kb of human genomic DNA.21 The transcript is encoded by five exons, and all exons contain the coding sequences indicated in Fig. 1A. In addition, exon V contains the 30 -untranslated region, which includes the polyadenylation signal AATAAA. Notably, the human genome also contains a related, processed pseudogene that covers approximately 1.4 kb of the genomic sequence and shares 95.3% nucleotide identity with exons 1–5 of GlcAT-I.21 The pseudogene is localized to chromosome 3. The mouse GlcAT-I locus is located on chromosome 19-A, is organized in a very similar fashion (Fig. 1B), and spans approximately 8.5 kb of the mouse genomic sequence. In contrast to the human genome, there is no GlcAT-I pseudogene in the mouse genome. So far, orthologues have been found at least in the zebrafish, Drosophila, and C. elegans genomes,22 suggesting the existence of conserved functions in different organisms.
III. cDNA and Protein Structure of GlcAT-I GlcAT-I activity was first detected in a chick embryo cartilage extract23 and was subsequently partially purified from embryonic chick brain24 and mouse mastocytoma cells25; however, attempts to purify GlcAT-I to homogeneity have not been successful due to its low concentration and difficulty in solubilizing the enzyme. Previously, the cDNA encoding a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope on glycoproteins (GlcAT-P) was cloned.26 GlcAT-P is a b1,3-glucuronyltransferase that utilizes glycoprotein acceptor substrates having the terminal Galb1-4GlcNAc
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A Human ATG
TGA ~3.2 kb //
Exon I
II
III
IV
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TGA ~4.0 kb //
Exon I
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8.5 kb 500 bp FIG. 1. Comparison of the genomic organization of human and mouse GlcAT-I. (A) Schematic illustration of the human GlcAT-I locus on chromosome 11q12.3. GlcAT-I gene consists of five exons and spans approximately 7 kb of human genomic sequence. (B) Schematic illustration of the mouse GlcAT-I locus on chromosome 19-A. GlcAT-I gene consists of five exons and spans approximately 8.5 kb of mouse genomic sequence. Its genomic organization is very similar to that of human GlcAT-I. Exon regions are denoted by boxes. Closed boxes represent the coding sequence, and open boxes denote 50 - and 30 -untranslated sequences. Translation initiation (ATG) and termination (TGA) codons are also shown. Black horizontal bars denote introns.
sequence. Mixed-substrate experiments have indicated that GlcAT-I and GlcATP are distinct enzymes, although the two enzymatic reactions are similar.27 In view of the resemblance in the reactions catalyzed by GlcAT-I and GlcAT-P, we cloned the cDNA encoding GlcAT-I from human placenta based upon information on the amino acid sequence alignment of rat GlcAT-P with the putative proteins in C. elegans and Schistosoma mansoni.17 Subsequently, based on the sequence homology to human GlcAT-I (GeneBank TM accession number AB009598), the mouse cDNA and genes were identified (GeneBank TM accession number AB019523). The human GlcAT-I cDNA had a single open reading frame of 1005-bp coding for a 335 amino acid protein showing the characteristics of a type II transmembrane protein, with one prominent hydrophobic segment of 18 amino acid residues in the N-terminal region, and a Golgi transmembrane domain.17,28 Mouse GlcAT-I cDNA also encodes a 355 amino acid, type II transmembrane protein that was 95% identical to human GlcAT-I. The overall fold of the catalytic domain (Thr-76-Val-335) of human GlcAT-I can be divided into two subdomains, the N-terminal cofactor binding domain and the C-terminal
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substrate-binding domain.29 Both human and mouse proteins contain a DDD sequence in their C-terminal, which appear to correspond to the conserved DXD motif found in most glycosyltransferases.30 One or two N-linked glycosylation sites are present in the C-terminal part of human and mouse proteins, respectively.17 The predicted size of the human or the mouse protein is approximately 37 kDa; however, both, human and mouse, recombinant protein A-tagged GlcAT-I showed two bands. Removal of the N-glycan from both the recombinant GlcAT-I proteins by enzymatic digestion resulted in the expected single band, suggesting that one potential N-linked glycosylation site of GlcAT-I is partly utilized.
IV. Enzymatic Activities GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine, Galb13Galb1-4Xylb1-O-Ser, thus finalizing the formation of the common linkage region, GlcAb1-3Galb1-3Galb1-4Xylb1-O-Ser. Although GlcAT-I was first identified in 1969, the properties of GlcAT-I were not described in detail until the cDNA encoding GlcAT-I was cloned.17,28 The properties and substrate specificities of recombinant GlcAT-I, which was expressed in COS-1 cells as a soluble, protein A chimera and purified using immunoglobulin G (IgG)Sepharose, have now been determined.18,28 Recombinant human GlcAT-I utilizes only a linkage region trisaccharide derivative, such as Galb1-3Galb1-4Xyl and Galb1-3Galb1-4Xylb1-O-Ser, and little GlcA incorporation is observed with other substrates containing a terminal GalNAc or Gal residue (i.e., polymer chondroitin), longer oligosaccharide– serines derived from the linkage region, N-acetyllactosamine, lactose, asialoorosomucoid, Galb1-3GlcNAc, Galb1-3GalNAc, and, notably, Galb1-3Galb1-Obenzyl.17,18 In addition, recombinant mouse GlcAT-I utilizes a linkage region trisaccharide derivative, such as Galb1-3Galb1-4Xyl. Thus, GlcAT-I is distinct from the enzyme termed glucuronyltransferase II, which is involved in the formation of the repeating disaccharide units of CS.11–13 The above mentioned substrate specificity indicates that the minimum structural requirement for the acceptor substrate of GlcAT-I is the trisaccharide sequence, Galb1-3Galb14Xyl (Km 80.4 mM), and that the enzyme recognizes up to the third saccharide residue (Xyl) from the nonreducing end.18 This deduced trisaccharide recognition disagrees with the finding of Wei et al.,28 who claimed that Galb13Galb1-O-napthalenemethanol and Galb1-3Galb1-O-benzyl served as acceptors of recombinant hamster GlcAT-I obtained by expression cloning. X-ray crystallographic analysis revealed the crystal structure of recombinant human GlcAT-I in the presence of both the donor-substrate product UDP and the acceptor substrate analog Galb1-3Galb1-4Xyl, and helped the identification of the key residues involved in catalysis and the binding of the two Gal residues.29
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The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively.31–36 Enzyme assays showed that the synthetic molecules, Galb1-3Galb1-4Xyl(2-O-phosphate)-O-Ser and Galb1-3Gal(6-Osulfate)b1-4Xyl(2-O-phosphate)-O-Ser, were better substrates for the truncated form of the recombinant human GlcAT-I than the unmodified trisaccharide serine, whereas Gal(6-O-sulfate)b1-3Galb1-4Xyl(2-O-phosphate)-O-Ser exhibited no acceptor activity.37 The crystal structure of the catalytic domain of GlcAT-I with UDP and bound Galb1-3Gal(6-O-sulfate)b 1-4Xyl(2-O-phosphate)-O-Ser revealed that the Xyl(2-O-phosphate)-O-Ser is disordered and that the 6-O-sulfate interacts with Gln-318 from the second GlcAT-I monomer in the dimeric enzyme.29,37 The above results indicate the possible involvement of such modifications in the processing and maturation of the growing linkage-region-oligosaccharide required for the assembly of GAG chains. In addition, the sulfotransferase responsible for the 6-O-sulfation of Gal residues and the kinase responsible for the 2-O-phosphorylation of the Xyl residue in the linkage region have been identified.38,39
V. Homologous Proteins GlcAT-I belongs to a group of Golgi-associated b1,3-glucuronyltransferases whose founding member is the human HNK-1 glucuronyltransferase.26,40,41 Phylogenetic analysis showed that GlcAT-I is closely related to the homologous proteins GlcAT-P and GlcAT-S. The amino acid sequence of GlcAT-I has a proline-rich domain (from Pro-30 to Pro-75) next to the transmembrane region, as is seen in several other glycosyltransferases, including GlcAT-P and GlcAT-S, which synthesizes the precursor structure GlcAb1-3Galb1-4GlcNAc-R for the HNK-1 carbohydrate epitope GlcA(3-O-sulfate)b1-3Galb1-4GlcNAc-R.26,40,41 Database searches indicate that the amino acid sequence of GlcAT-I displays 43% and 46% sequence identity to GlcAT-P and GlcAT-S, respectively. The highest sequence identity is found in the C-terminal catalytic domain, which follows the proline-rich region (252 amino acids between Pro-68 and Glu-319 overlap with about 60% identity) 17,28 and contains the four previously identified highly conserved motifs (I–IV) of putative glucuronyltransferases among animal species.26,39
VI. Expression Pattern of GlcAT-I Northern blot analysis for GlcAT-1 mRNA demonstrated a single band of 1.5 kb in all human tissues examined.21 The GlcAT-I gene exhibited ubiquitous but differential expression in the human tissues examined. Notably,
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expression was particularly abundant in the placenta, followed by the pancreas, brain, and heart. In addition, Northern blot analysis of mouse mRNA demonstrated a single band of 1.8 kb (Fig. 2). The mouse GlcAT-I gene was expressed from embryonic day 7 (Fig. 2B). In adult tissues, its expression was detected in virtually all the tissues examined, with the highest levels found in the liver, brain, and kidney (Fig. 2A). These expression patterns of mouse GlcAT-I have also been previously described.28 Additionally, to establish the expression pattern of GlcAT-I during mouse embryogenesis, in situ hybridization was carried out. At embryonic day 8, GlcAT-I was expressed in the telencephalon, hindbrain, and in the frontonasal prominence. At embryonic day 16, GlcAT-I was prominently expressed in the teeth, thymus, kidney, and cartilage primordia (unpublished results). These results indicate a highly specific temporal and spatial expression pattern of GlcAT-I during embryogenesis.
VII. GlcAT-I in Mouse Early Embryogenesis A. Targeted Disruption of GlcAT-I Results in Early Embryonic Lethality
2.4
7
5
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kb
E1
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E7 E1
kb 4.4
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ea r Br t ai Sp n le Lu en ng Li ve Sk r el Ki eta dn l m e us Te y cl e st is
To examine the functions of GlcAT-I in mammalian early embryogenesis, the GlcAT-I gene was inactivated via homologous recombination in mouse ES cells.20 The targeting vector was constructed by inserting a neomycin resistance cassette into exon II (Fig. 3).
4.4 2.4
1.35 1.35 FIG. 2. Northern blot analysis of mouse GlcAT-I. Northern blots with mRNAs from various mouse tissues (A) or embryonic stages (B) were hybridized with a probe for mouse GlcAT-I cDNA. (A) Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. (B) Lane 1, embryonic day 7; lane 2, embryonic day 11; lane 3, embryonic day 15; lane 4, embryonic day 17.
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SmaI
SmaI
6.4 kb
//
// Exon I 7.0 kb
EcoRI
Exon I //
II
III
Exon I
V
Wild-type allele
EcoRI
II NEO
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//
IV
EcoRI
Targeting vector
DT-A SmaI
8.0 kb II NEO
III
IV //
Targeted allele
1 kb EcoRI
8.6 kb
EcoRI
FIG. 3. Targeted disruption of mouse GlcAT-I gene.20 The neomycin resistance cassette was inserted into exon II of GlcAT-I gene. Coding and noncoding exons of GlcAT-I gene are shown by closed and open boxes, respectively, and the PGKneobpA cassette (NEO) and diphtheria toxin A fragment gene cassette (DT-A) are represented by gray boxes.
GlcAT-Iþ/– mice had an apparently normal phenotype and were born at a largely Mendelian frequency. They were intercrossed and more than 300 offspring were genotyped by PCR. No GlcAT-I–/– neonates and embryos after E6.5 could be detected, indicating that the mutant embryos died during early embryogenesis (Table I). In fact, even at E2.5 (8-cell stage), only 2% of the GlcAT-I/ embryos were detected, suggesting that most GlcAT-I/ mutants died before E2.5 (8-cell stage).20
B. GlcAT-I is Essential for Embryonic Cytokinesis and Cell Division To further analyze the lethalilty and phenotypes of the GlcAT-I–/– embryos during early development, 2-cell stage embryos, derived from heterozygous intercrosses (GlcAT-Iþ/ GlcAT-Iþ/) or other matings (GlcAT-Iþ/ GlcATIþ/þ or GlcAT-Iþ/þ GlcAT-Iþ/þ), were cultured until the blastocyst stage (Table II). The results of the in vitro culture experiments showed that 13% of the embryos from the GlcAT-Iþ/ heterozygous intercrosses died between the 2-cell and 8-cell stage, although all embryos from GlcAT-Iþ/ GlcAT-Iþ/þ or GlcAT-Iþ/þ GlcAT-Iþ/þ were viable (Table II). Notably, of the embryos from
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
27
TABLE I GENOTYPE ANALYSIS OF PROGENY FROM GLCAT-I HETEROZYGOUS INTERCROSSES20 No. of progeny with genotypea Day
þ/þ
þ/–
–/–
No. resorbedb
No. total
Neonate
46 (38%)
74 (62%)
0 (0%)
–
120
E8.5
22 (39%)
31 (56%)
0 (0%)
3 (5%)
56
E7.5
12 (19%)
43 (67%)
0 (0%)
9 (14%)
64
E6.5
17 (39%)
18 (41%)
0 (0%)
9 (20%)
44
E2.5
19 (45%)
22 (53%)
1 (2%)
–
42
Percentages of different genotypes appear in parentheses. a Genotyping of each developmental stage was performed by PCR. b Resorbed embryos were not genotyped.
the heterozygous intercrosses, only 7% GlcAT-I/ embryos could be identified at the implantation stage, while the fraction of the GlcAT-Iþ/þ and the GlcATIþ/ embryos that were viable was within Mendelian expectations (1:2), confirming that GlcAT-I inactivation is lethal before the 8-cell stage. Moreover, reversion of cell division was observed in embryos only from the GlcAT-Iþ/ heterozygous intercrosses. Figure 4 shows a representative example of the reversion of cell division in one embryo from the GlcAT-Iþ/ heterozygous intercrosses. The 2-cell (E1.5) embryo divided into a 4-cell embryo, and then insufficient cytoplasmic division seemed to force the embryonic cell compartments to revert to an unusual 3-cell embryo with four nuclei. This unusual embryo eventually died, most likely due to incomplete cytokinesis (Fig. 4A and B). These results indicate that GlcAT-I function is essential for embryonic cytokinesis and cell division.
C. Expression of CS and HS in Mouse Embryos As mentioned earlier, because GlcAT-I transfers GlcA from UDP-GlcA to the trisaccharide-serine, Galb1-3Galb1-4Xylb1-O-Ser, thus finalizing the formation of the common GAG–protein linkage region GlcAb1-3Galb1-3Galb14Xylb1-O-Ser,17 it was expected that inactivation of GlcAT-I would abolish both CS and HS chains in mouse embryos. Immunocytochemistry with wild-type mouse 2-cell embryos and blastocysts was first performed using an anti-CS (LY111) or anti-HS (Hepss-1) monoclonal antibodies. As expected, fluorescent signals were detected in all 2-cell embryos (Fig. 5C and D) and blastocysts with
TABLE II GENOTYPE ANALYSIS OF EMBRYOS CULTURED IN VITRO FROM 2-CELL-STAGE EMBRYOS TO BLASTOCYST IMPLANTATION STAGES20 No. of progeny with genotypea
No. of dead embryosb
8-cell to morula
Morula to blastocyst
Blastocyst to hatched blastocyst
Total dead embryos
No. total
Parental genotype
þ/þ
þ/–
–/–
2-cell to 8-cell
GlcAT-Iþ/ GlcAT-Iþ/
32 (29%)
55 (49%)
8 (7%)
15
0
0
2
17 (15%)
112
GlcAT-Iþ/ GlcAT-Iþ/þ
23 (59%)
14 (36%)
–
0
0
0
2
2 (5%)
39
GlcAT-Iþ/þ GlcAT-Iþ/þ
42 (98%)
–
–
0
1
0
0
1 (2 %)
43
Percentages of different genotypes appear in parentheses. a Genotyping was performed by PCR. b Dead embryos were not genotyped, but their lethal stages were determined.
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
A
E1.5
B
E2.0
E2.5
29
E3.0
Hoechst
FIG. 4. Reversion of cytokinesis in embryos from GlcAT-I heterozygous intercrosses.20 (A) E1.5 embryos were isolated from heterozygous crosses and cultured. Representative features are depicted. Reversal of cytokinesis was observed in one embryo from GlcAT-I heterozygous intercrosses (arrowhead). No reversal of cytokinesis was detected in embryos from crosses of wild-type and heterozygous mice. (B) The two embryos shown in panel (A) were stained with Hoechst 33342. The abnormal embryo (arrowheads in panel (A)) failed to complete cytokinesis and a double nucleated cell appeared (upper), whereas cell division of the other embryo continued normally (lower).
either of these antibodies; and the corresponding signals were eliminated by chondroitinase ABC (CSase) or heparitinases (HSase), respectively, indicating that both CS and HS were produced in mouse 2-cell embryos and blastocysts.20
D. Inactivation of GlcAT-I Results in Defective CS and HS Next, double immunostaining of GlcAT-I/ and GlcAT-Iþ/ embryos as well as GlcAT-Iþ/þ embryos using anti-CS (LY111) and anti-DHS (3G10) monoclonal antibodies was carried out. GlcAT-Iþ/þ and GlcAT-Iþ/ embryos were stained by both anti-CS and anti-DHS monoclonal antibodies, whereas the GlcAT-I/ embryos were not stained.20 These findings suggest that GlcATI/ embryos seem to lack both the CS and HS chains.
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IZUMIKAWA AND KITAGAWA
A
C
LY111
B
D
Hepss-1
FIG. 5. Immunocytohistochemistry of mouse 2-cell embryos using an anti-CS or anti-HS monoclonal antibody.20 Wild-type embryos at E1.5 were collected and stained. Left panels (A, B) show embryos examined by phase-contrast microscopy. Immunofluorescent staining by anti-CS (LY111) or anti-HS (Hepss-1) antibody is shown in right panels (C, D).
E. CS Chains are Involved in Controlling Embryonic Cell Division and Cytokinesis As described above, GlcAT-I/ embryos showed loss of synthesis of both CS and HS and died before the 8-cell stage due to failure of cytokinesis. However, it is not clear whether the embryonic cell death observed in the GlcAT-I/ embryos is due to deficiency of CS, HS, or both. If CS or HS is indispensable for proper embryonic cytokinesis and cell division, digestion of CS or HS at the embryonic cell surface might also induce abnormal cell division. The treatment of 2-cell embryos with CSase showed that 67% of treated embryos died between the 2-cell and 8-cell stages. In contrast, most embryos treated with heat-inactivated CSase/HSase and 65% of embryos treated with HSase developed normally in to blastocysts (Table III, Fig. 6). These results indicate that CS chains, but not HS chains, are involved in controlling embryonic cell division and cytokinesis.
VIII. Concluding Remarks It has been revealed that in C. elegans, nonsulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, while HS is essential for embryonic morphogenesis in the later stages of development.2–4 In addition, prior studies on EXT1 or EXT2 in mice demonstrated that
IMPAIRMENT OF EMBRYONIC CELL DIVISION IN GLUCURONYLTRANSFERASE-I
31
TABLE III ANALYSIS OF LETHAL STAGES OF IN VITRO CULTURED EMBRYOS AFTER TREATMENT WITH CSASE OR HSASE20 No. of dead embryos
Controla
2-cell to 8-cell
8-cell to morula
Morula to blastocyst
No. of viable embryos to blastocyst stage
No. total
0
0
2
29
31
CSase treatment
22
5
0
6
33
HSase treatment
2
5
1
15
23
a
Embryos were treated with heat-inactivated CSase/HSase.
E1.5
E2.5
E2.75
E3.5
E4.0
Control
A
CSase
B
HSase
C
FIG. 6. Depletion of CS results in cytokinesis defects.20 Wild-type embryos at E1.5 were collected and incubated with heat-inactivated CSase/HSase (A, control), CSase (B), or HSase (C), respectively. Treatment of embryos with CSase showed that these two embryos died from 2-cell to 8-cell stages (B), although control (A) and HSase-treated (C) embryos developed normally to blastocysts. Representative features are depicted.
these genes are essential for HS synthesis and early development.5,6 Notably, EXT1- or EXT2-null embryos developed normally until around E6.5, when they became growth-arrested and failed to gastrulate. In addition, the marked reduction of HS in ES cells from EXT1- or EXT2-deficient mice has been reported.5,6 To clarify the roles of CS in early embryogenesis in mammals, GlcAT-I knockout mice were generated by gene targeting.20 Mice with a deletion of GlcAT-I showed marked reduction of the synthesis of CS and HS and embryonic lethality before the 8-cell stage due to failed cytokinesis.
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In addition, treatment of wild-type 2-cell embryos with CSase had marked effects on cell division, although many HSase-treated embryos normally developed to blastocysts. These results suggest that CS in mammals, as with nonsulfated chondroitin in C. elegans, is indispensable for embryonic cell division. So far, there are no reports on the role of CS or CSPG in cytokinesis and cell division in mice. To gain more insight into the role of CSPG in mammalian cell division, future studies on the identification of core proteins modified with CS, that are involved in mouse embryonic cell division, are needed. Moreover, although the GlcAT-I gene is ubiquitously expressed in virtually every human and mouse tissue examined, it exhibits marked differential expression among tissues.17,28 Thus, conditional knockout of the gene in mice would provide essential information regarding the biological function of sulfated GAGs in each tissue.
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