Glycobiology Advance Access originally published online on May 19, 2008
Glycobiology 2008 18(8):615-625; doi:10.1093/glycob/cwn042
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POMT2, a key enzyme in Walker–Warburg syndrome: somatic sPOMT2, but not testis-specific tPOMT2, is crucial for mannosyltransferase activity in vivo
Department of Cell Chemistry, Heidelberg Institute of Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany
1 To whom correspondence should be addressed: Tel: +49-(0)6221-546286; Fax: +49-(0)6221-545859; e-mail: sstrahl{at}HIP.uni-heidelberg.de
Received on April 8, 2008; revised on May 9, 2008; accepted on May 12, 2008
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Abstract |
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O-Mannosylation represents an evolutionarily conserved, essential protein modification. In mammals the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. Mutations in human POMT1 and POMT2 cause a group of congenital muscular dystrophies due to reduced O-glycosylation of
-dystroglycan. The most severe of these autosomal recessive conditions is Walker–Warburg syndrome (WWS) with severe brain and ocular involvement. We previously showed in the murine model that Pomt1 is expressed in WWS-related tissues both during embryogenesis and in adults. Whereas there is only a single Pomt1 transcript in adult mice, we demonstrated that there are two Pomt2 transcripts, somatic sPomt2 and testis-specific tPomt2. In this study we demonstrate that sPomt2, but not tPomt2, is prominently expressed in mouse embryos in the tissues that are most severely affected in WWS (developing muscle, eye, and brain). Correlation of POMT transcripts and protein isoforms with POMT mannosyltransferase enzyme activity demonstrates that sPOMT2–POMT1 complexes catalyze mannosyltransfer in adult somatic tissues and testis. It is suggested that the gonadal defects described in some WWS cases are associated with defects in O-mannosylation. Our data further show that whereas sPOMT2 is widely expressed, tPOMT2 is restricted to the acrosome of male germ cells and is not involved in the biosynthesis of O-mannosyl glycans in vivo. We prove that tPOMT2 is highly conserved among mammals, including humans, suggesting a crucial function that is distinct from sPOMT2. Key words: glycosylation / mannosylation / mannosyltransferase / POMT2 / POMT1
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Protein O-mannosylation is a conserved modification in eukaryotes and some bacteria. Defects in the assembly of O-mannosyl glycans are of fatal consequences in diverse organisms ranging from yeasts to humans (reviewed in Lehle et al. 2006
)
The biosynthesis of O-mannosyl glycans is initiated in the endoplasmic reticulum (ER) by the transfer of a mannose residue from dolichyl phosphate-activated mannose (Dol-P-Man) to specific serine or threonine residues of proteins entering the secretory pathway (reviewed in Willer et al. 2003). Further assembly of O-mannosyl glycans occurs in the Golgi apparatus where additional sugar residues are gradually transferred from nucleotide-activated sugar donors. In mammals the vast majority of O-mannosyl glycans represent variations of the tetrasaccharide NeuAc2-3Galβ1-4GlcNAcβ1-2Man-Ser/Thr; these structures vary in length and in fucose content (Willer et al. 2003).
The initiation of O-mannosylation in the ER is catalyzed by members of the essential PMT-family of dolichyl phosphate-D-mannose:protein O-mannosyltransferases that was first identified in yeast and is conserved throughout the animal kingdom with the exception of worms (Strahl-Bolsinger et al. 1993; Gentzsch and Tanner 1996; Willer et al. 2003). In mammals two PMT-family members have been identified, namely POMT1 and POMT2 (Jurado et al. 1999; Willer et al. 2002). Analogous to the yeast system complex formation between POMT1 and POMT2 proteins has been demonstrated, and it has been shown that complex formation is crucial for mannosyltransferase enzyme activity in vitro (Manya et al. 2004).
POMT1 and POMT2 are essential for mammalian development. Targeted disruption of Pomt1 in mice results in embryonic lethality (Willer et al. 2004). In humans mutations in either POMT1 or POMT2 are associated with a broad clinical spectrum of autosomal recessive congenital muscular dystrophies (CMD) such as Limb-girdle muscular dystrophy type 2K and 2N (LGMD2K and LGMD2N; Balci et al. 2005; Biancheri et al. 2007) and neuronal migration disorders such as Walker–Warburg syndrome (WWS; Beltran-Valero de Bernabe et al. 2002; van Reeuwijk et al. 2005) and muscle–eye–brain disease (MEB; Mercuri et al. 2006). Varying degrees of CMD, brain malformation (cobblestone lissencephaly) with mental retardation, and abnormalities of the eye are characteristics of these neuromuscular disorders (reviewed in Muntoni et al. 2004). Diseases related to defects in POMT1 or POMT2 are also referred to as secondary dystroglycanopathies. The key feature of this genetically heterogeneous group is a defect in the posttranslational modification (hypoglycosylation) of -dystroglycan which is so far the only well-characterized O-mannosylated protein in mammals and humans (reviewed in Barresi and Campbell 2006). Due to multiple severe malformations, WWS patients often die within the first year of life. In POMT-deficient patients with the MEB or LGMD phenotype, however, the disease progresses less rapidly and some patients can reach adulthood (Balci et al. 2005; Mercuri et al. 2006). In addition to neuromuscular defects, severe malformations of male gonads have been described in several WWS cases (Dobyns et al. 1989; Hung et al. 1998) suggesting that O-mannosyl glycans are also crucial for the development of testis. This assumption is further supported by the finding that male infertility is observed in mice lacking protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which catalyzes the first elongation step of O-mannosyl glycans in the Golgi apparatus (Liu et al. 2006).
In mice during embryonic development Pomt1 is expressed in brain, muscle, and eye which is consistent with the neuronal, muscle, and eye abnormalities found in WWS patients (Willer et al. 2004; Prados et al. 2007). In adult rodents and humans POMT1 is expressed in all tissues investigated; however, expression is somewhat elevated in testis (Willer et al. 2004; Manya et al. 2006). Compared to POMT1 considerably less is known about POMT2. We showed that similar to POMT1 in adult mice and humans POMT2 is expressed in all tissues but expression is predominant in testis (Willer et al. 2002, 2004). Interestingly, contrary to Pomt1 in mice two distinct transcript species that vary in length are formed due to differential transcription initiation of the Pomt2 gene. The shorter transcript (somatic Pomt2; sPomt2) is present in all tissues examined. The longer transcript (testis Pomt2; tPomt2) is highly abundant in testis and encodes a predicted testis-specific tPOMT2 protein isoform bearing 70 additional amino acids (aa) at its N-terminus (Willer et al. 2002). Immunohistochemical localization using antibodies that do not discriminate the POMT2 isoforms showed that in testis cross-sections of adult mice POMT2 localizes to both nonspermatogenic and spermatogenic cells but is predominant in the acrosome of maturing spermatids (Willer et al. 2002), a sperm-specific organelle that is indispensable for successful fertilization (Abou-Haila and Tulsiani 2000). A testis-specific tPomt2 transcript was also observed in rat (Manya et al. 2006). No direct evidence has as yet been reported that the tPOMT2 protein occurs in species other than rodents.
In this study we show that in mouse embryos sPomt2 is prominently expressed in the neural tube, the developing eye, and the mesenchyme. A detailed characterization of Pomt2 in mouse testis proves that sPOMT2, but not tPOMT2, can act as a mannosyltransferase in vivo in complex with POMT1. We demonstrate that tPOMT2 is conserved in mammals and humans, affirming a crucial role for the testis-specific POMT2 isoform.
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Results |
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To learn more about the primary features of POMTs we performed a detailed characterization with a focus on POMT2 using mouse as a model system.
sPomt2 is expressed in WWS-related tissues during embryogenesis
To monitor Pomt2 expression during mouse embryonic development we performed Northern blot analysis of different developmental stages. In embryos, as previously reported for adult tissues, two sPomt2 transcripts at 2.8 kb and 4.7 kb are expressed due to differential polyadenylation (Willer et al. 2002; Figure 1A). The expression levels of sPomt2 are similar at all developmental stages analyzed. The 3.1 kb testis-specific tPomt2 transcript (Willer et al. 2002) was not detected demonstrating that only sPomt2 is expressed during embryogenesis.
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Furthermore whole-mount in situ hybridizations on mouse E10.5 embryos revealed that low-level expression of sPomt2 mRNA is relatively ubiquitous. Higher levels of sPomt2 expression are observed in the neuronal tissues. Transcripts are found throughout the neural tube and the developing trigeminal ganglion (Figure 1B). In addition, sPomt2 is expressed in the somites, the limb-bud mesenchyme, and the developing eye (Figure 1B). sPomt2 expression highly resembles the Pomt1 expression pattern that we previously described (Willer et al. 2004
), and sites of expression correlate with those in which the main tissue alterations are observed in WWS and MEB patients.
In adult tissues sPOMT2 levels correlate with POMT mannosyltransferase activity
In adult mice Pomt2 mRNA is expressed in all tissues but expression is predominant in testis (Willer et al. 2002). In our previous experiments Pomt2 expression was evaluated by Northern blot hybridization. To have a more accurate estimate of the expression, we performed quantitative RT-PCR to quantify Pomt2 mRNA compared to Pomt1 and PomGnt1 in adult tissues. In all somatic tissues analyzed, only slight variations of the expression levels of each of the three genes are found. Relatively low expression of Pomt1, Pomt2, and PomGnt1 mRNA is detected in muscle (Figure 2A). Compared to muscle, mRNA levels are increased 
2-fold in brain and 5-fold in kidney and liver. Interestingly, in testis the expression pattern is different. Although Pomt1 and PomGnt1 mRNA levels in testis largely resemble expression in liver, Pomt2 mRNA levels in testis are increased 7-fold relative to liver and 50-fold relative to muscle (Figure 2A).
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Since POMT1 and POMT2 act in a complex to catalyze O-mannosyltransferase activity (Akasaka-Manya et al. 2006
), the salient increase of Pomt2 expression in testis was unexpected. To relate mRNA expression with enzymatic activity we measured POMT mannosyltransferase activity in microsomal membranes isolated from mouse testis, brain, muscle, kidney, and liver tissue. The highest POMT enzyme activity is detected in kidney and liver (Figure 2B) whereas POMT activity in testis is not much different from that in muscle and brain tissue and is 2-fold lower than in kidney and liver tissue. Therefore POMT enzyme activity correlates with Pomt1 and not with Pomt2 mRNA expression (Figure 2A). The data suggest that the strong increase in Pomt2 mRNA expression in testis is not connected to enzymatic activity.
Our previous work demonstrated that in mouse testis Pomt2 transcripts encode two isoforms, namely sPOMT2 and tPOMT2, the latter featuring a putative 70-aa N-terminal extension (Willer et al. 2002). To directly assess the two POMT2 isoforms we performed Western blot analyses of microsomal membranes isolated from testis and liver using antibodies that are directed against a central domain (val-373 to leu-470) of human POMT2 (anti-POMT2; Figure 4A) and recognize both sPOMT2 and tPOMT2 of a mouse. Similar quantities of sPOMT2 (apparent Mr of 81 kDa) are detected in testis and liver (Figure 2C, lanes 1 and 2). However, tPOMT2 (apparent Mr of 87 kDa) is highly abundant in testis and exceeds the amount of sPOMT2 8-fold, demonstrating that the strong increase in Pomt2 transcripts in testis (Figure 2A) is due to the induction of tPomt2 expression. Taking into consideration that O-mannosyltransferase activity is not enhanced in testis (Figure 2B) our data show that sPOMT2 but not tPOMT2 contributes to POMT activity in testis in vivo.
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The N-terminal extension does not influence localization and membrane topology of tPOMT2 in vitro
To investigate the impact of the testis-specific N-terminal extension we first characterized tPOMT2 in vitro. Therefore, we created C-terminally hemagglutinin (HA)-tagged versions of sPOMT2 and tPOMT2 and expressed those in 293 human embryonic kidney (HEK) cells. Western blots of microsomal fractions prepared from those cells decorated with monoclonal anti-HA antibodies showed that both POMT2HA isoforms are stably expressed (supplementary Figure 1). Indirect immunolocalization of sPOMT2HA and tPOMT2HA revealed a specific and strong perinuclear and reticular staining for both isoforms (Figure 3). Signals overlap with the ER marker protein ERp72 demonstrating ER localization of both POMT2 isoforms in vitro.
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Our previous work showed that yeast protein O-mannosyltransferase ScPmt1p is an integral ER membrane protein with seven transmembrane domains (Strahl-Bolsinger and Scheinost 1999
). The N-terminus faces the cytoplasm, whereas two major loop regions (loops 1 and 5) and the C-terminus are situated in the lumen of the ER. Based on the high similarity of the hydropathy profiles within yeast and mammalian PMT-family members, this topology model is applicable to POMT2 (Figure 4A). To analyze whether the N-terminal extension affects the orientation of tPOMT2 in the ER membrane we performed indirect immunofluorescence studies using different antibodies that specifically recognize distinct domains of tPOMT2HA (Figure 4A). Fibroblast cells were fixed and membranes selectively permeabilized with 5 µg/mL digitonin (specific for the plasma membrane) or 0.2% Triton X-100 (permeabilization of plasma and internal membranes). Control staining for cytosolic tubulin and the ER-resident protein calreticulin confirmed the selectivity of the permeabilization procedure (Figure 4B). Staining with antibodies directed against the hydrophilic domains loop 1 (anti-loop1), loop 5 (anti-POMT2), and the C-terminus (anti-HA), respectively, was only achieved upon permeabilization of both the plasma and ER membrane thereby proving the lumenal orientations of these regions of the protein (Figure 4B). Summarizing, in vitro the tPOMT2-specific N-terminus affects neither ER localization nor overall membrane topology of tPOMT2 according to our topological model.
tPOMT2 is restricted to the acrosome of spermatogenic cells and mature sperm
To further analyze POMT2 isoforms in vivo we raised polyclonal antibodies (anti-tPOMT2) against an 18-aa peptide that corresponds to Thr-15 to Arg-32 of the mouse tPOMT2 N-terminal extension (Figure 8D). Specificity of anti-tPOMT2 was confirmed using HA-tagged versions of the mouse POMT2 isoforms expressed in fibroblast cells (supplementary Figure 1). Further, Western blot analyses of microsomal membranes isolated from mouse testis and liver demonstrated that anti-tPOMT2 exclusively recognizes the 87 kDa tPOMT2 protein in testis (Figure 2C, lanes 3 and 4).
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Using anti-POMT2 antibodies we previously established that in testis POMT2 is present in haploid germ cells as well as in nonspermatogenic cells such as Sertoli and Leydig cells (Willer et al. 2002
). To define the precise distribution of the two POMT2 isoforms we performed immunohistological analyses using anti-tPOMT2 antibodies. Immunostaining on cross-sections of mouse seminiferous tubules revealed a selective and strong positive staining. Preadsorption of anti-tPOMT2 with the respective blocking peptide resulted in the complete ablation of immunoreactivity (data not shown). Asymmetrical accumulation of the anti-tPOMT2 signal (Figure 5A; brown color) on one side of the nucleus in the capping phase and more advanced elongated spermatids demonstrates that tPOMT2 localizes to the acrosome. No signal is observed in spermatocytes and spermatogonia of different meiotic maturation stages. The anti-tPOMT2 staining in haploid germ cells of different spermiogenic stages highly resembles the staining pattern that we observed using anti-POMT2 antibodies which do not discriminate between the POMT2 isoforms (Willer et al. 2002). However, different from anti-POMT2, anti-tPOMT2 does not stain Sertoli and Leydig cells (Figure 5A; Willer et al. 2002). To further define those differences we increased the sensitivity of immunostaining using Alexa Fluor 488-conjugated secondary antibodies. Both anti-POMT2 and anti-tPOMT2 antibodies selectively stain the acrosome of haploid spermatocytes (Figure 5B; green color) but the staining of Leydig cells is exclusively observed with non-testis-specific anti-POMT2.
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Taken together our immunohistological analyses prove that in testis both POMT2 isoforms are stably expressed. However, in contrast to tPOMT2, which is restricted to developing spermatogenic cells, sPOMT2 is present in nonspermatogenic cells.
We further monitored the occurrence and localization of tPOMT2 during acrosome biogenesis. The acrosome is formed during spermiogenesis and represents a defining feature of sperm development in testis (Abou-Haila and Tulsiani 2000). According to acrosome formation spermiogenesis is divided in four distinct phases: Golgi, cap, acrosome, and maturation phase (Moreno and Alvarado 2006). During the Golgi phase proacrosomal granules fuse to form a single large acrosomal vesicle. During the cap phase, the spherical acrosomal vesicle is enlarged. Finally during acrosome and maturation phase, the acrosomal vesicle becomes hemispherical and the acrosome is formed. To follow tPOMT2 localization spermatogenetic germ cells were isolated by the enzymatic dissociation of adult mouse testis. Spermatocytes and postmeiotic spermatids were fixed, permeabilized, and stained with either anti-POMT2 or anti-tPOMT2 antibodies to distinguish the POMT2 protein isoforms. Acrosomal status was monitored by tetramethyl rhodamine isothiocyanate (TRITC)-coupled peanut agglutinin (PNA) an acrosomal marker lectin (Burkett et al. 1987). Both POMT2 antibodies reveal diffuse staining in spermatocytes that resembles ER staining (Figure 6A and B). At the onset of spermiogenesis POMT2 staining cumulates in the acrosomal vesicle (Figure 6A and B, Golgi phase), remains tightly associated with the maturing acrosome in the course of spermiogenesis, and persists to spermatzoa. In contrast to anti-tPOMT2 staining, which is largely restricted to the developing acrosome (Figure 6B), significant anti-POMT2 staining is also observed in the cellular body of maturing spermatids at all stages of sperm development (Figure 6A), indicating that tPOMT2 but not sPOMT2 specifically localizes to the acrosome. Further, analysis of mouse epidydimal sperm showed that the acrosomal tPOMT2 staining persists in mature sperm (Figure 6D).
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Colocalization studies suggest that in embryonic (Willer et al. 2004
; Figure 1) and adult somatic tissues (Prados et al. 2007) sPOMT2–POMT1 complexes are formed in vivo. To explore putative tPOMT2–POMT1 complexes in testis in vivo we analyzed POMT1 localization during spermatogenesis using anti-POMT1 antibodies. As shown in Figure 6C, in contrast to tPOMT2, POMT1 staining is predominantly found in the ER of developing germ cells and is even observable in the cytoplasmic droplet of spermatozoa that contains the remains of the ER (Figure 6C). Accumulation of POMT1 in the developing acrosome occurs, but not until the maturation phase (Figure 6C, maturation), and maintains in spermatozoa. In summary, immunofluorescence analyses of POMTs in developing spermatids and mature sperm show that in testis sPOMT2 and POMT1 are mainly located in the ER of nonspermatogenic cells and developing spermatids whereas tPOMT2 exclusively localizes to the acrosome, suggesting a distinct role for tPOMT2 in vivo.
tPOMT2 is conserved in mammals
We previously showed that mouse tPomt2 transcripts contain an additional in frame ATG-start codon 210 base pairs (bp) upstream of the somatic start codon (Figure 7 and Willer et al. 2002). To analyze whether tPOMT2 is also present in other mammalian species we screened various mammalian genomes for mouse sPOMT2 homologues using TBLASTN (Altschul et al. 1997). 5'-sequences of the sPOMT2 genes identified were then searched for additional ATG codons that could give rise to tPOMT2. In addition to rodents, putative tPOMT2 isoforms were identified in cat, dog, bovine, and horse (Figure 7). Qualitative analysis of these tPOMT2 ATG-start codons with respect to their ability to serve as translation start (NetStart1.0; Pedersen and Nielsen 1997) revealed that in all cases translation initiation is highly likely. Moreover, predictions favor the earlier start codons with respect to the somatic ATG start. Among all species analyzed the 210 bp sequence between the two ATG-starts is highly conserved (Figure 7) and the corresponding protein sequences share 53–94% identity and 60–94% similarity.
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To confirm the general occurrence of tPOMT2 in mammals we analyzed membrane proteins from bovine testis and brain by Western blot. Anti-POMT2 antibodies recognize both sPOMT2 and tPOMT2 in bovine tissues (Figure 8A, lanes 1 and 2). As in mouse, bovine sPOMT2 shows an apparent Mr of
81 kDa whereas tPOMT2 migrates with an apparent Mr of 87 kDa. In contrast, anti-tPOMT2 exclusively recognizes the 87-kDa tPOMT2 protein in testis (Figure 8A, lanes 3 and 4). In summary, our data demonstrate that tPOMT2 is not only present in rodents but is also evolutionary conserved among mammals.
tPOMT2 is conserved in humans
Also in humans the 5'-genomic sequence upstream the sPOMT2 ATG-start is highly conserved. However, a 1 bp deletion at position bp –182 causes a frame shift between the putative tPOMT2 and the sPOMT2 ATG-starts (Figure 7). Interestingly splicing of a putative intron between bp –74 and bp –1 could neutralize the frame shift mutation. The corresponding tPOMT2 transcript should encode the tPOMT2 protein containing a 45-aa N-terminal extension with high homology to the mammalian sequence (Figure 8D). To address this question we used a PCR-based approach and analyzed human POMT2 transcripts in testis and blood. Thereto, total RNA of the respective tissues was reverse transcribed and the corresponding cDNA analyzed as described in Materials and methods. The presence of hPOMT2 transcripts in both tissues was confirmed using PCR primers directed against the somatic coding region (Figure 8C, primer pair tw41/ml63, and B, lanes 1 and 2). Next a forward primer (Oligo411) within the putative intronic sequence was combined with primer ml63 to specifically detect nonspliced cDNA variants. The resulting 298 bp PCR product is detected in cDNA derived from blood (Figure 8B, lane 4) as well as from testis (Figure 8B, lane 3) indicating the presence of a transcript population bearing the 74 bp intron sequence in both tissues. However, only a minor fraction of the hPOMT2 cDNA in testis is detected (Figure 8B, compare lanes 1 and 3) suggesting the presence of a spliced transcript variant. To clarify whether splicing of the putative intron between bp –74 and bp –1 of the hPOMT2 transcript occurs, a primer combination (primer pair tw186/ml63) that encompasses the suspected sequence was selected. As shown in Figure 8B (lanes 5 and 6) both the spliced (315 bp PCR product) and the nonspliced (389 bp PCR product) transcript variants are detected in testis whereas in blood only the nonspliced transcript is traced. To verify that the human tPOMT2 transcript includes the testis-specific ATG-start codon PCR reactions were performed using primer ml63 in combination with primer tw213 and E1f1, respectively. Transcripts comprising the tPOMT2 start codon were detected only in testis (Figure 8B, lanes 7 and 9) but were absent in blood (Figure 8B, lanes 8 and 10).
Our analyses demonstrate that in humans splicing of a 74 bp intron compensates a single base deletion in the tPomt2-specific coding region. The resulting transcript encodes a truncated, but highly conserved tPOMT2 isoform suggesting that tPOMT2 is also crucial in humans.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Mutations in human POMT1 and POMT2 are the proximate cause for CMD associated with clinical heterogeneity (Godfrey et al. 2007
). In this study we show that in the murine model during embryogenesis sPomt2 is strongly expressed in the developing muscle, eye, and brain (Figure 1). The observed expression pattern highly correlates with expression sites of Pomt1 (Willer et al. 2004) and dystroglycan (Anderson et al. 2007), the only known major substrate of POMTs. Since POMT1–POMT2 complexes are required to achieve protein O-mannosyltransferase activity in vitro (Akasaka-Manya et al. 2006) our data signify that also in vivo POMT1–sPOMT2 complexes are crucial for the development of tissues affected in WWS and other dystroglycanopathies. In adult somatic tissues however, relative expression of sPomt2 and Pomt1 is lowest in muscle and brain in agreement with low POMT activity in the respective tissues (Figure 2). Elevated levels of Pomt1 and Pomt2 expression and POMT activity are observed in liver and kidney (Figure 2). Different to embryos in adult tissues levels of POMT activity do not correlate with reported expression levels of dystroglycan that is high in skeletal muscle but low in liver and kidney (Bedossa et al. 2002). The presence of alternative, yet unidentified substrates of protein O-mannosyltransferases in the respective tissues might explain these discrepancies.
Our previous analyses revealed that in contrast to somatic tissues, a testis-specific tPomt2 transcript is expressed in spermatocytes of different meiotic stages (Willer et al. 2002) which often transcribe genes whose products will be specifically used later to form the acrosome (Tanaka and Baba 2005). Using tPOMT2-specific antibodies we directly prove the existence of the tPOMT2 protein (Figure 2C). Immunolocalization shows that tPOMT2 is exclusively present in spermatogenic cells (Figure 5). At the onset of acrosome formation tPOMT2 is targeted to the developing acrosome and localization in the acrosomal membrane persists in mature sperm (Figure 6). In contrast, sPOMT2 is also present in nonspermatogenic cells such as Leydig cells (Figure 5), resembling the distribution of POMT1 (Prados et al. 2007). Throughout spermatide maturation sPOMT2 and POMT1 show predominant ER localization and only a minor fraction of POMT1 is present in the acrosome of elongated spermatids but not before the maturation phase (Figure 6). From our data we conclude that sPOMT2 and tPOMT2 serve distinct purposes in testis tissue.
Colocalization of sPOMT2 and POMT1 in testicular tissue (Willer et al. 2002; Prados et al. 2007) and developing sperm (Figures 5 and 6) suggests the presence of enzymatic active POMT1–sPOMT2 complexes in vivo in the ER of nonspermatogenic and spermatogenic cells that are involved in the biosynthesis of O-mannosyl glycans. The functional role of O-mannosyl glycans in testis is not defined. But gonadal defects have been described in at least three WWS cases (Hung et al. 1998), and in mice lacking POMGnT1, an enzyme that catalyzes the elongation of O-mannosyl glycans, male infertility is observed (Liu et al. 2006). The gonadal or rather testicular deficiencies of WWS patients and POMGnT1 knock-out mice are not investigated thoroughly. Thus, it has to be elucidated whether these defects arise out of hypoglycosylation of -dystroglycan, which appears to be limited to the basement membrane of seminiferous tubules (Durbeej et al. 1998) or other yet unknown O-mannosylated proteins.
Different to sPOMT2, tPOMT2 is targeted to the acrosome of spermatids and mature sperm independent of POMT1 (Figure 6), where it might serve a function not related to the enzymatic activity due to the following reasons. First, to our knowledge the presence of Dol-P-Man, the mannosyl donor substrate of POMTs, is limited to the ER (Schenk et al. 2001). Second, although the enzymatic activity of rat tPOMT2 has been demonstrated in cell culture (Manya et al. 2006), the pH optimum of the transfer reaction is at pH 8.5 whereas at pH 5.8, the average intra acrosomal pH value (Nakanishi et al. 2001), almost no activity is detected (Manya et al. 2004). In cell culture tPOMT2 is associated with the ER (Figures 3 and Figure 4); thus, in vitro tPOMT2 mannosyltransferase activity does not reflect the in vivo situation. Finally, the comparison of POMT mannosyltransferase activity of different mouse tissues revealed that enzymatic activity in testis does not resemble the high expression level of tPomt2 mRNA in that tissue (Figure 2). POMT activity is even lower in testis compared to brain, liver, or kidney where exclusively sPomt2 is expressed. However, POMT activity in testis correlates well with the level of the sPOMT2 protein (Figure 2C) suggesting that in testis only ER-located POMT1–sPOMT2 complexes but not tPOMT2 accomplish the mannosyltransfer reaction.
We proved that tPOMT2 is highly conserved between mammals and humans (Figures 7 and 8). Protein BLAST searches of the nonredundant protein sequence database using the 70-aa N-terminal extension of tPOMT2 revealed no significant homology to any known protein suggesting that this domain is a unique feature of tPOMT2 proteins. In contrast to other mammals, in primates including humans the genomic sequence in between the tPOMT2 and sPOMT2 ATG-start shows a 1 bp deletion (Figure 7 and data not shown). This mutation is compensated for by splicing an intron in immediate proximity to the sPOMT2 start (Figure 8). The corresponding human tPOMT2 protein contains a 45-aa N-terminal extension with high homology to the mammalian sequence (Figure 8) and is located in the acrosome of human sperm (data not shown). The high degree of conservation of tPOMT2 throughout the animal kingdom and human suggests a crucial role in the acrosome.
To address tPOMT2 function we searched for putative protein interaction sites within the N-terminal tPOMT2-specific peptide. Using the eukaryotic linear motif resource (Puntervoll et al. 2003; http://elm.eu.org/about.html) we identified SH3- and PDZ-binding motifs as well as Cyclin-1 and MAPK-1 interacting motifs (data not shown). In addition, we identified proteins that interact with the tPOMT2-specific N-terminal extension peptide via a yeast two-hybrid screen. Among several putative tPOMT2 interacting proteins the disheveled-2 protein was identified (M.L. and S.S., unpublished data). Disheveled proteins are known to interact with ligands such as Frizzled through their PDZ domains and are as part of the Wnt-signaling pathway involved in several differentiation processes. Furthermore, for the disheveled-2 homolog, disheveled-1, a functional role during testis development has been proposed (Ma et al. 2006). Although further work will be necessary to establish the functional link between tPOMT2, disheveled-2, and other interacting proteins our data suggest that in the acrosome tPOMT2 fulfils a function independent of mannosyltransferase activity.
Ectopic localization of Golgi glycosyltransferases is frequently found in various tissues (Berger 2002). The most likely function of ectopic glycosyltransferases is related to their intrinsic carbohydrate-binding specificities. An interesting example is β1,4-galactosyltransferase (GalT) that normally serves as a biosynthetic enzyme in the Golgi apparatus; however in sperm it also functions as a cell–surface receptor for extracellular glycoside ligands (reviewed in Rodeheffer and Shur 2002). Due to differential transcription initiation a testis-specific GalT isoform is made that is located in the plasma membrane of sperm where it functions as a receptor for the major sperm-binding ligand on the extracellular coat of the egg. The testis-specific N-terminal domain of GalT is involved in the activation of intracellular signaling pathways after sperm–egg contacts have occurred (Shi et al. 2001).
Even if the role of the testis-specific tPOMT2 isoform is not clear at the moment, its features and high degree of conservation between mammals and humans point to a lectin-like role during acrosome maturation or fertilization which remains to be elucidated in the future.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Multiple sequence alignments were generated with CLUSTAL W (Thompson et al. 1994
). Hydropathy profiles were generated using ProtScale (Gasteiger et al. 2005) with a 17-aa window.
Northern blot analysis
A mouse embryo multiple tissue Northern Blot (Clontech, Mountain View, USA) was hybridized according to the manufacture's instruction with 32P-labeled Pomt2 (bp 360–690) as described previously (Willer et al. 2002).
Whole-mount in situ hybridization
Embryos from timed pregnancies were dissected in PBS and fixed overnight at 4°C in 4% paraformaldehyde/PBS. Sense and antisense Pomt2 probes (bp 360–690) were prepared using a digoxigenin RNA labeling kit (Roche Diagnostics, Basel, Switzerland). Whole-mount in situ hybridizations were carried out as described (Willer et al. 2004).
Cell lines and culture
Wild-type 293 HEK cells were cultured in Dulbecco modified Eagle's medium supplemented with 10% fetal calf serum, 60 µg/ mL penicillin, and 100 µg/mL streptomycin. Transfected cells were selected and maintained in 200 µg/mL hygromycin (Roche Diagnostics). Cell lines were cultivated at 37°C in a 5% CO2 incubator.
Antibody production
Anti-tPOMT2 antiserum was produced using a synthetic peptide corresponding to aa Thr-15 to Arg-32 of the N-terminal extension of mouse tPOMT2. Anti-Loop1 antibodies were generated using a peptide corresponding to aa Thr-126 to Lys-141 of mouse sPOMT2. For coupling to keyhole limpet hemocyanin (KLH) a cystein residue was added to the C-terminus of the peptide. KLH-peptide conjugates were injected into rabbits. Antibodies were affinity purified using the respective peptide coupled to cyanogen bromide-activated sepharose. Peptide synthesis, coupling to KLH, immunizations, and affinity purifications were done at Pineda Antiköer Service (Berlin).
tPOMT2 and sPOMT2 expression constructs
Mouse testis cDNA was obtained by the reverse transcription of mouse total testis RNA (Clontech) using an Omniscript reverse transcriptase kit (Qiagen, Hilden, Germany) and oligo-dT primers following manufacturer's instructions.
Plasmid pTW55.
To obtain mouse sPomt2HA the coding sequence was amplified by PCR with the primer pair tw179 (ccgCTCGAGgatgccgccggccataggcggt XhoI site is underscored)/tw180 (ataagaatGCGGCCGCcaaagtcccatgattccagccac, NotI site is underscored) using mouse testis cDNA as a template. To fuse three copies of the HA epitope to the C-terminus of sPomt2 the 2.3 kb PCR fragment was cloned as XhoI/NotI fragment into pREP3x-tag (Willer et al. 2005) cut with the same enzymes.
Plasmid pCEP4
KpnI.
To delete the KpnI-restriction site in the mammalian expression vector pCEP4 (Invitrogen, Carlsbad, USA), the plasmid was digested with KpnI, 3'-protruding ends were removed and the plasmid was religated.
Plasmid pML10.
sPomt2HA was excised from pTW55 as a 2.4 kb XhoI/SmaI fragment and ligated into pCEP4KpnI which was cut with BamHI, 3'-recessed ends were filled-in and subsequently cut with XhoI. The resulting plasmid pML10 carries sPomt2HA under the transcriptional control of the CMV promoter.
Plasmid pML11.
To obtain mouse tPomt2HA a 730 bp fragment carrying the 210 bp 5'extended sequence of tPomt2 plus 520 bp of the somatic coding sequence was amplified by PCR using the primer pair ml1 (5'-ttcccAAGCTTctcccgcaatgctctacg-3', HindIII site is underscored)/tw71 (5'-ggacttggacagatccagtac-3'). The PCR product was cut with HindIII/KpnI and subcloned into pML10. The resulting plasmid pML11 carries tPomt2HA under the transcriptional control of the CMV promoter.
Isolation of microsomal membrane fractions
Mircosomal membrane fractions from cultured cells or mouse tissues were isolated as described previously (Manya et al. 2006).
Western blot analysis
Microsomal membrane fractions from fibroblasts (50 µg protein) or different tissues (80–240 µg protein) were separated on 8% sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose. As primary antibodies affinity-purified polyclonal anti-tPOMT2 (1:100), anti-POMT2 (1:500; Willer et al. 2002), and monoclonal anti-HA antibodies (1:5000, Babco, Richmond, USA; 16B12) were used. After decoration with secondary anti-rabbit (1:5000, Sigma, St. Louis, USA) or anti-mouse (1:5000, Sigma) antibodies coupled to HRP, immunoreactivity was visualized by enhanced chemiluminescence using the Pierce SuperSignal West Pico substrate.
Assay for protein O-mannosyltrasferase activity
Protein O-mannosyltrasferase activity was determined based on the amount of [3H]-mannose transferred from Dol-P-[3H]-mannose to an -dystroglycan glutathione-S-transferase fusion as described elsewhere (Manya et al. 2004).
Analysis of human testis transcripts
One microgram total RNA (Clontech) from either testis or whole blood was reverse transcribed using an iSelect reverse transcription kit (BioRad, Hercules, USA). PCR reactions were performed using the primers E1f1 (5'-agaccagtggcc- agcttgatgtc-3'), tw213 (5'-ccggtcccgacatgctctgc-3'), tw186 (5'-cagtgaagggcgttgcatttcc-3'), Oligo 411 (5'-gccggagggcga- cccagaggag-3'), and tw41 (5'-atgccgccggccacgggcgg-3'), respectively, in combination with primer ml63 (5'-ccaaagtga- gtctcatcccaacagat-3'). One microliter RT reaction was used in each reaction as a template.
Quantitative expression analysis in mouse tissues
Two microgram of total RNA from different mouse tissues were reverse transcribed using an iSelect reverse transcription kit (BioRad). PCR reactions were performed in the iCycler iQ real-time polymerase chain reaction detection system (BioRad) using iQ Sybergreen Supermix (BioRad). Each reaction contained cDNA derived from 20 ng RNA as a template and 100 nM of gene-specific primers (primer sequences are summarized in supplementary Table I). Transcript levels were normalized to Ppia and Hprt1 as described elsewhere (Vandesompele et al. 2002).
Immunohistology
Immunohistochemical studies were carried out as described previously (Willer et al. 2002). Primary antibodies were applied in PBS/normal blocking serum at 1:25 (anti-tPOMT2) and 1:100 (anti-POMT2) dilutions overnight at 4°C. Anti-rabbit-biotin (1:200) secondary antibodies were applied in PBS/normal blocking serum for 1 h at room temperature. In the case of immunofluorescence analyses, the ABC-reaction was replaced by incubation with Alexa Fluor 488-conjugated avidin (1:1000, Molecular Probes, Carlsbad, USA) diluted in PBS/normal blocking serum for 30 min at room temperature. Nuclei were visualized with 0.2 µg/mL DAPI (Calbiochem, Darmstadt, Germany) in PBS. Sections were mounted in a ProLong Antifade reagent (Molecular Probes) and cured overnight. Specificity of anti-tPOMT2 antibodies was demonstrated by preadsorbing the primary antibodies with the respective peptide-epitope, replacing the primary antibodies by preimmune serum, and its substitution with a buffer.
Isolation of spermatogenic cells
Cell suspensions of seminiferous epithelium were prepared from adult mice by sequential enzymatic digestion as described elsewhere (Bellve et al. 1977). Cells were allowed to adhere to poly-L-lysine (Sigma)-coated microscopic slides and fixed for 5 min in 100% methanol at –20°C. Thereafter slides were immediately used for immunofluorescence analysis.
Selective permeabilization of cultured cells
293 HEK cells were grown on poly-lysine-coated cover slips for 1 day. Cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, and incubated for 20 min in 50 mM NH4Cl in PBS. Selective permeabilization of the plasma membrane was achieved by incubation in the buffer containing 5 µg/mL digitonin, 0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl2, 1 mM EDTA, and 10 mM HEPES pH 6.9 for 15 min at 4°C as previously described (Eckhardt et al. 1999). Complete permeabilization was achieved by incubation in PBS containing 0.2% Triton X-100 for 30 min at room temperature.
Immunofluorescense analysis of spermatogenic cells and cultured cells
Nonspecific binding sites were blocked in 3% BSA/PBS. Thereafter slides were incubated with either polyclonal anti-tPOMT2 (1:25), polyclonal anti-POMT2 (1:250), polyclonal anti-loop1 (1:100), polyclonal anti-POMT1 (Prados et al. 2007; 1:500), monoclonal anti-tubulin antibodies (1:1000, Sigma), monoclonal anti-calreticulin antibodies (1:250, Calbiochem), polyclonal anti-ERp72 antibodies (1:1000, Calbiochem), monoclonal anti-HA antibodies (1:1000, Babco, 16B12), and polyclonal anti-HA antibodies (1:500, Santa Cruz Biotechnologies Inc.) diluted in PBS containing 1% BSA overnight at 4°C. Secondary Alexa Fluor 488-conjugated anti-mouse (1:1000, Molecular Probes) or Cy3-conjugated anti-rabbit (1:250, JacksonImmuno Research, West Grove, USA) were diluted in PBS/1% BSA. Acrosomes of spermatogenic cells were stained with 10 µg/mL TRITC-conjugated Arachis hypogaea agglutinin (PNA; Sigma) in PBS/1% BSA supplemented with 0.5 mM CaCl2 and 0.5 mM MgCl2. Nuclei were stained with 0.2 µg/mL DAPI in PBS. Between all incubation steps cells were washed three times with PBS. After a final washing step, cells were mounted with a ProLong Antifade kit (Molecular Probes) and cured overnight.
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Supplementary Data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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This work was supported by the Deutsche Forschungs Gemeinschaft (grant STR443/2).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank R. Donhauser, C. Endres, and A. Metschies for excellent technical assistance. We are grateful to J. Cruces for generously providing anti-POMT1 antibodies. We thank the members of our lab for many helpful discussions.
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Footnotes |
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2 Present address: Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52240, USA.
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Abbreviations |
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aa, amino acid; bp, base pair; CMD, congenital muscular dystrophies; DAPI, 4',6-diamdino-3-phenylindol; Dol-P-Man, dolichyl phosphate-activated mannose; ER, endoplasmic reticulum; GalT, β1,4-galactosyltransferase; HA, hemagglutinin; 293-HEK cells, 293-human embryonic kidney cells; KLH, keyhole limpet hemocyanin; LGMD, limb-girdle muscular dystrophy; MEB, muscle–eye–brain disease; PNA, conjugated Arachis hypogaea agglutinin; POMGnT1, protein O-mannose 1,2-N-acetylglucosaminyltransferase1POMT,PMT, protein O-mannosyltransferase; sPomt2, somatic Pomt2; tPomt2, testis-specific Pomt2; TRITC, tetramethyl rhodamine isothiocyanate; WWS, Walker–Warburg syndrome
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