2 Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany; 3 Institut für Anatomie und Physiologie der Haustiere, Universität Hohenheim, 70593 Stuttgart, Germany; and 4 Lehrstuhl für Biochemie, Universität Regensburg, 93040 Regensburg, Germany
Received on May 18, 2002; revised on July 5, 2002; accepted on July 9, 2002
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Abstract |
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Key words: glycosylation/mannosyltransferase/PMT/POMT/testis
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Introduction |
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Until recently, O-mannosylation was considered to be a yeast-specific phenomenon (Gemmill and Trimble, 1999). However, over the past few years it has become clear that among mammalian proteins O-linked mannose is not as uncommon as it was previously believed (Endo, 1999
; Yuen et al., 1997
; Smalheiser et al., 1998
). Different from fungi, in mammals all O-mannosyl glycan structures elucidated so far are variations of the tetrasaccharide Sia
2-3Galß1-4GlcNAcß1-2Man-Ser/Thr variegating in length and/or containing fucose of different linkage arrangements (Endo, 1999
). A limited number of O-mannosylated glycoproteins from brain, nerve, and skeletal muscle, such as the extracellular matrix receptor
-dystroglycan, has been described (Endo, 1999
). A recent study demonstrated that muscle-eye-brain disease (MEB), an autosomal recessive disorder in humans, is caused by a defect in O-mannosyl glycan synthesis and proposes a new pathomechanism for muscular dystrophy and neuronal migration disorders (Yoshida et al., 2001
).
In yeasts and fungi the synthesis of O-mannosyl linked carbohydrate chains is initiated in the ER by a conserved family of dolichyl phosphate-D-mannose:protein O-mannosyltransferases (Pmt), which catalyze the transfer of a mannosyl residue from dolichyl phosphateactivated mannose to specific serine/threonine residues of proteins entering the secretory pathway (Strahl-Bolsinger et al., 1999). In S. cerevisiae, a total of seven PMT family members (ScPmt17p) have been identified. Characterization of the PMT family members of S. cerevisiae revealed a signature seven-transmembrane helical structure that distinguishes Pmt proteins from all other glycosyltransferases (Strahl-Bolsinger and Scheinost, 1999
). Phylogenetic analysis indicates that the protein O-mannosyltransferases fall into three major subfamilies, PMT1, PMT2, and PMT4 subfamily, and include transferases closely related to S. cerevisiae ScPmt1p, ScPmt2p, and ScPmt4p, respectively (Girrbach et al., 2000
; this study). In spite of a high degree of conservation, clear distinctions exist between PMT1/PMT2 and PMT4 subfamily members, including certain intrinsic features of the amino acid sequence and pronounced specificity toward protein substrates (Girrbach et al., 2000
; Gentzsch and Tanner, 1997
). Furthermore, in yeast the PMT1 and PMT2 but not the PMT4 subfamily is highly redundant (Girrbach et al., 2000
).
So far all known O-glycosylation reactions are vastly different in yeast compared to those of higher eukaryotes (Gemmill and Trimble, 1999). Therefore, it was surprising when PMT4 subfamily members were identified from Drosophila melanogaster (rotated abdomen, rt), and from human (hPOMT1, protein O-mannosyltransferase) (Martin-Blanco and Garcia-Bellido, 1996
; Jurado et al., 1999
). Drosophila rt mutants feature reduced viability as well as pronounced defects in muscle development, indicating an essential role of PMTs in not only lower but also higher eukaryotes. However, to date besides their functional importance very little is known about PMT homologs from higher eukaryotes.
In this study we present the cloning of the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). Northern and western blot analyses of the mammalian POMT2, with emphasis on mouse POMT2, revealed that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene two mRNAs are produced that differ in length. The longer of the two is restricted to testis and encodes a testis-specific isoform of the mPOMT2 protein. Immunological analyses demonstrate that the mPOMT2 protein localizes to and is abundant within the acrosome of maturing spermatids. Our results suggest a specific role for the mammalian putative protein O-mannosyltransferase POMT2 during spermiogenesis and/or fertilization.
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Results |
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A genomic clone of mouse Pomt2 (mP2g3: ICRFP703O24280Q5) was isolated by screening a genomic filter library (Lib. No.: 703/RZPD) with a 449-bp polymerase chain reaction (PCR) fragment amplified on EST clone IMAGE:1181688. Clone mP2g3 served as template to determine the genomic sequence of the mouse Pomt2 gene by a PCR-based strategy. PCR fragments making up the intron sequences were amplified with cDNA-derived primers, subcloned, and sequenced. Alignments of the genomic and the cDNA sequences pinpointed intronexon junctions, which were confirmed by the existence of predicted splice signals strictly observing the AG/GT rule (Mount, 1982). Analogous to the hPOMT2 gene, the mouse Pomt2 transcription unit consists of 21 exons spanning a genomic region of 40.9 kb (Genbank Accession No. AY090483; Figure 2). Although the mouse Pomt2 gene has not been mapped to a chromosome yet, it is interesting to note that the human chromosomal region 14q24 is of conserved synteny with mouse chromosome 12 (LocusID: 184986).
Considered together, the complete human POMT2 cDNA from cerebellum covers a sequence of 4.9 kb and the mouse Pomt2 cDNA from liver a sequence of 4.7 kb. In exon 21 both human and mouse cDNAs are variable in length by 2.0 kb due to alternative polyadenylation (Figure 1). Human and mouse POMT2 genes encode proteins of 750 aa that are 91% identical (Figure 1). Furthermore, the genomic organization is conserved between the human and mouse POMT2 gene (Figure 2).
Cloning of the Drosophila POMT2 homolog cDNA
A TBLASTN (Altschul et al., 1997) search of the Drosophila EST database with the amino acid sequence of the mammalian POMT2 protein identified the 2.6-kb cDNA clone LP01681 (Genbank Accession No. AI258474), encoding a predicted protein with high homology to human and mouse POMT2. Sequence analysis of clone LP01681 revealed a unique ORF of 2295 bp encoding a predicted protein of 765 amino acids. Mouse and fly POMT2 proteins feature an overall sequence identity of 54% and, accordingly, a similarity of 64%. In contrast to the mammalian POMT2, the fly homolog shows an additional stretch of 47 amino acids (aa 609655) that is absent within all other PMT family members (Figure 3). A BLASTP search of the Genbank database did not reveal any significant homology between this 47-aa sequence and any previously defined gene or protein. We confirmed that these additional 47 codons are not due to incomplete splicing of clone LP01681 by amplifying cDNA fragments that cover the coding sequence of aa 493689 (by reverse transcription [RT]-PCR on mRNA of total flies) and subsequent sequence analysis (data not shown). Furthermore, semiquantitative RT-PCR showed that the fly POMT2 gene is expressed in both fly heads and bodies with a slightly higher expression level observed in heads (data not shown).
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Features of the mammalian and fly POMT2 proteins
Conceptual translation of the mammalian and fly POMT2 cDNAs revealed highly hydrophobic proteins of 750 and 765 aas, respectively, containing five (human), six (mouse), and seven (Drosophila) N-glycosylation sites. Their hydropathy profiles are typical of putative protein O-mannosyltransferases of the PMT family and predict a seven-transmembrane helical structure with a large central hydrophilic domain flanked by five amino- and two carboxyl-terminal transmembrane spans (Figure 4A) (Strahl-Bolsinger and Scheinost, 1999). Like all other known PMT family members, POMT2 lacks typical ER-targeting and ER-retention signals. A comparison of the POMT2s with proteins of the Genbank, Swissprot, and PIR databases confirmed their assignment to the PMT family. POMT2s and yeast Pmt-proteins feature an overall sequence similarity of 4246% and identity of 3337%. Mammalian POMT2 and the previously described human POMT1 protein (Jurado et al., 1999
) share 46% similarity and 36% identity; the Drosophila POMT2 homolog and Drosophila rt protein (Martin-Blanco and Garcia-Bellido, 1996
) share 43% similarity and 33% identity.
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Mammalian POMT2 is an integral membrane protein of the ER membrane
In contrast to the vast majority of protein glycosyltransferases acting in the Golgi apparatus, PMTs initiate protein O-mannosylation in the ER in yeasts (Strahl-Bolsinger et al., 1999). Because the subcellular localization of PMTs in higher eukaryotes is unsettled, we wished to determine whether the mammalian POMT2 protein localizes to the ER. To investigate its intracellular localization biochemically as well as immunologically, we fused three copies of the hemagglutinin (HA) epitope to the C-terminus of human cerebellum POMT2 cDNA (hPOMT2HA), which includes the ATG-start codon at position bp 485 (see Figure 1). We stably transfected a human 293 kidney fibroblast cell line with hPOMT2HA under the control of the constitutive cytomegalovirus promoter. Cells were fractionated into soluble and crude membrane fractions, and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) and western blot. For immunological detection we used monoclonal anti-HA antibodies and affinity-purified polyclonal anti-POMT2 antibodies, which were raised in rabbits (see Materials and methods).
Figure 5A shows that the hPOMT2HA protein is stably expressed in fibroblast cells. The endogenous level of hPOMT2 protein appeared to be very low, because no hPOMT2 protein could be detected in untransfected fibroblast cells with anti-POMT2 antibodies, which recognize hPOMT2HA very specifically (Figure 5A, lane 4). Cell fractionation experiments showed that hPOMT2HA fractionated completely with the membrane fraction (Figure 5A, compare lanes 2 and 5 and lanes 3 and 6). On SDSpolyacrylamide gels the protein migrates with an apparent molecular weight (MW) of approximately 83 kDa. Treatment with endoglycosidase H (Endo H) indicates that at least two out of five potential N-glycosylation sites are used in vivo (Figure 5B). Removal of all N-linked carbohydrate chains results in an apparent MW of 77 kDa. The discrepancy between the detected MW of 77 kDa and the calculated MW of 88 kDa can be explained by an aberrant migration behavior due to the very hydrophobic nature of the hPOMT2 protein and was also observed for other PMT family members (Girrbach et al., 2000).
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Expression of the mammalian POMT2 gene is most prominent in testis
In addition to the subcellular localization, we were interested in the tissue specificity of mammalian POMT2. The distribution of human and mouse POMT2 mRNA in different tissues was examined by northern blot analyses. A RNA Master blot (Clontech) containing mRNA from 18 different mouse adult tissues and different control RNA and DNA samples was analyzed using a murine Pomt2 exon 25-specific probe. mPomt2 is expressed in all tissues investigated, but the level is about sevenfold higher in testis (Figure 6A). A very similar expression pattern was obtained for the human POMT2 gene in human tissues (Figure 6A). In addition, we analyzed a mouse multiple-tissue northern blot (MTN; Clontech). In seven out of eight different murine tissues (heart, brain, spleen, lung, liver, skeletal muscle, and kidney) two distinct mPomt2 transcripts of 2.7 kb and 4.7 kb specifically hybridized with the exon 25-specific probe (Figure 6B, upper panel). Similar quantities of the two transcripts were detected. 3'-RACE revealed that the size variation of the transcripts is due to alternative polyadenylation (Figure 1). Notably, expression of mPomt2 in testis is about fivefold higher than in the other tissues (Figure 6B, upper panel), and surprisingly, in addition to marginal amounts of the 2.7 kb and 4.7 kb mRNAs, two dominant transcripts are present that are slightly longer (3.1 kb and 5.1 kb). Further, polyadenylation at position bp 3076 seems to be preferred (Figure 6B, upper and middle panel).
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Due to the earlier transcription start site, two distinct mPomt2 mRNA species emerge, the longer being restricted to testis. Analysis of the testis-specific mPomt2 5'-sequence uncovered that besides several very short ORFs, an ATG-start codon is present at position bp 275, which is in frame with the mPomt2 ATG-start codon of exon 1 at position 485 bp (Figure 1). Consequential an extended mPomt2 ORF originates coding for a deduced testis-specific mPOMT2 protein isoform of 820 aa (Genbank Acc. AY090481; Figure 1). Qualitative analysis of the different ATG-starts with respect to the Kozak rule (Kozak, 1992) favor the earlier ATG-start at position 275 bp as translation start site. Taken together, our data show that the mPomt2 gene is expressed in all tissues investigated but predominantly in testis. Due to differential transcription initiation, two distinct mPomt2 mRNA species varying in length are produced. The shorter transcript is found in all tissues investigated. The longer transcript is specifically present in testis tissue and might encode a putative mPOMT2 protein isoform featuring a 70-aa extension of the N-terminal region (Figure 1).
In testis mPOMT2 protein is localized predominantly to the acrosome of maturing spermatids
To further exploit the association of mPOMT2 protein with testis, we isolated crude membranes from various mouse adult tissues, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. Membrane proteins were resolved on 8% SDSpolyacrylamide gels and analyzed by western blot using polyclonal anti-POMT2 antibodies. Because anti-POMT2 antibodies are directed against the central hydrophilic domain of human POMT2 (valine-373 to leucine-470), which is 94% identical between the human and the mouse protein, they specifically recognize both human and mouse POMT2 protein (Figures 5 and 7). As shown in Figure 7A, we could verify mPOMT2 protein particularly in testis tissue, confirming our northern data. The testis mPOMT2 protein migrates on SDSpolyacrylamide gels with an apparent MW of 87 kDa. Endo H treatment showed that as was found for human POMT2 protein, at least two out of six mPOMT2 N-glycosylation sites are used in vivo (data not shown). After raising the limit of detection of the western analysis (see Materials and methods) we could satisfactorily detect mPOMT2 also in membranes isolated from tissues other than testis, such as liver (Figure 7B). Interestingly, on SDSpolyacrylamide gels liver mPOMT2 protein shows a lower apparent MW when compared to testis mPOMT2 (~81 kDa versus 87 kDa). The discrepancy in MW of approximately 6 kDa strongly suggests that in testis the first translation initiation site is used (Figure 1; ATG-start at position bp 275) giving rise to a testis-specific mPOMT2 protein isoform that contains a 70-aa extension of the N-terminal region.
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The localization of mPOMT2 to the acrosome was further confirmed by indirect immunofluorescence staining of the mPOMT2 protein in testicular spermatids isolated from mouse testes (see Materials and methods). A mixture of postmeiotic spermatids of different developmental stages was fixed, permeabilized, and labeled with affinity-purified polyclonal anti-POMT2 antibodies followed by FITC-conjugated goat anti-rabbit secondary antibodies. In elongated spermatids of the late maturation phase mPOMT2 protein could be detected specifically in the acrosome (Figure 9B). Acrosomal localization was confirmed by colocalization of the mPOMT2 protein with the lectin peanut agglutinin (PNA), a marker for the sperm acrosome (Figure 9C, D) (Burkett et al., 1987). In contrast, in premeiotic spermatogenic cells where the acrosome is not yet present, a minor mPOMT2 signal is detected in the ER (data not shown). In summary, our data show that mPomt2 is specifically expressed during spermatogenesis. Transcription of mPomt2 mRNA is first observed in pachytene spermatocytes, whereas the protein can first be detected in round spermatids. In the latter, mPOMT2 localizes to the acrosome, a sperm-specific organelle.
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Discussion |
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Sequence searches within the public databases reveal a large number of PMT family members within all yeasts and fungi in general. In fungi the PMT family is highly redundant, ranging from seven to three members in S. cerevisiae and S. pombe, respectively (Figure 4B) (Strahl-Bolsinger et al., 1999; Willer et al., unpublished data). Fungal protein O-mannosyltransferases catalyze the first step in the synthesis of O-linked manno-oligosaccharides (Strahl-Bolsinger et al., 1999
). Work on S. cerevisiae Pmt enzymes showed that the individual family members recognize very specific protein substrates in vivo (Gentzsch and Tanner, 1997
; Mrsa et al., 1997
).
In higher eukaryotes the PMT family is less redundant compared to fungi. Because sequencing of the Drosophila and human genome is completed (Adams et al., 2000; Lander et al., 2001
), we are positive about that only one member of the PMT2 and PMT4 subfamily respectively, is present at a time in Drosophila, human, and mouse (Martin-Blanco and Garcia-Bellido, 1996
; Jurado et al., 1999
; this study). A BLAST search in the public EST database retrieved several PMT-homologous ESTs from rat, cattle, pig, dog, chicken, zebrafish, pufferfish and frog, indicating that PMTs (in vertebrates named POMTs) are widespread among higher eukaryotes. However, despite their common distribution the function of POMTs in these organisms is still an open question. Even though we employed a series of diverse experimental approaches, we have failed to detect in vitro protein O-mannosyltransferase activity of POMTs in vertebrates (Willer et al., unpublished data).
To prove a yeast-like O-mannosyl transfer reaction we used membrane fractions isolated from different organisms and tissues, such as testes (bovine), brain (rabbit), skeletal muscle (mouse; C2C12 myoblasts), or human 293 fibroblast cells expressing hPOMT2HA (Figure 5A) as an enzyme source. Dolichyl phosphateactivated mannose as well as GDP-mannose served as sugar donors. Furthermore, different mannose acceptors, such as endogenous membrane proteins (Marriott and Tanner, 1979), synthetic peptides, and specific proteins, were used. Among those, we tested 15 different synthetic peptides derived from
-dystroglycan (an O-mannosylated protein in vertebrates) and varying protein fragments of
-dystroglycan, which were expressed and purified as gluthatione S-transferase (GST) fusion proteins from Escherichia coli. However, we were not able to demonstrate a specific O-mannosyl transfer reaction under any conditions we tried. Yet this is not completely unexpected, taking in account that for most of the yeast Pmt enzymes no in vitro activity could be detected in spite of O-mannosyltransferase activity that can be detected in vivo (Gentzsch and Tanner, 1996
, 1997; Mrsa et al., 1997
; Willer and Strahl, unpublished data). This fact is attributed to the high substrate specificity of these enzymes (Gentzsch and Tanner, 1997
). In addition, compared to fungi where O-mannosylation is an abundant modification of many cell wall proteins, mammalian O-mannosyl glycosylation is a rare type of protein modification (Endo, 1999
). Thus, mammalian POMTs might catalyze a rather poor O-mannosyl transfer reaction, further complicating its detection in vitro.
We further addressed the question whether POMTs act as O-mannosyltransferases by functional complementation of yeast pmt mutants. However, heterologous expression of fly and human POMTs in S. cerevisiae and S. pombe pmt mutant strains, respectively, did not complement functional deficiencies (Willer and Strahl, unpublished data). Again, this is not surprising because in the course of these analyses it turned out that even between yeast species functional complementation of defects in protein O-mannosylation is difficult to achieve. Therefore, only through indirect evidence, summarized later, can we infer that POMTs are involved in the synthesis of O-mannosyl glycans.
The Drosophila rt mutant, which is defective in a putative protein O-mannosyltransferase of the PMT4 subfamily, produces a muscular dystrophylike phenotype with clockwise rotation of the abdomen (Martin-Blanco and Garcia-Bellido, 1996). A similar phenotype is observed for the Drosophila mutant twisted (tw) (FlyBase ID: FBgn0003898) which is caused by a mutation of the here newly assigned fly POMT2 homologous gene (Cruces, personal communication) (Davis, 1980
; Demerec et al., 1942
). Both Drosophila mutants show, depending on the allele an up to 90° clockwise rotation of the abdomen attended by severe defects in muscle development (Martin-Blanco and Garcia-Bellido, 1996
). Additionally, the mutants feature reduced fertility and viability.
In mammals POMT mutants have not yet been identified. However, a function for O-mannosyl glycans has been established in the case of the ubiquitously expressed -dystroglycan where the O-mannosyl linked sugar moiety was shown to be involved in binding of the extracellular matrix protein laminin (Chiba et al., 1997
). It is noteworthy that
-dystroglycans in different species and different tissues all have O-mannosyl glycans (Endo, 1999
). Therefore, it is postulated that O-mannosylation is a common feature for the
-dystroglycanlaminin interaction in multicellular organisms and that this forms a functional linker between the intracellular cytoskeleton and the extracellular matrix (Winder, 2001
). As indicated by the phenotypes of MEB, a disorder caused by a defect in the synthesis of O-mannosyl glycans, this function is very critical for a proper muscle development in mammals (Yoshida et al., 2001
; Kano et al., 2002
). Considering that the MEB-associated phenotypes in mammals are due to defects in the synthesis of O-mannosyl glycans and the similar phenotypes observed in Drosophila rt and twisted mutants, it seems highly likely that POMTs initiate protein O-mannosylation in higher eukaryotes. However, presently it cannot be excluded that POMTs may in fact transfer not mannose but a different sugar like glucose or fucose (Spiro, 2002
). Because neither a POMT mouse model is available nor a human disease is mapped to the genomic position of one of the POMT genes, the proposition that these genes encode O-mannosyltransferase activity in vivo will require substantiation from future studies. Our findings that mammalian POMT2 in nongerm cells localizes to the ER, like the yeast Pmt mannosyltransferases, and is expressed in many somatic tissues further support the idea that POMTs might catalyze a yeast-like mannosyl-transfer reaction in higher eukaryotes.
For mammalian POMT2 we observed a broad tissue expression pattern with high abundance in testis (Figures 6 and 7). A very similar expression profile was reported for the human POMT1 gene, indicating that in vertebrates POMTs fulfill an important and maybe specific function in testis (Jurado et al., 1999). In all somatic tissues investigated the transcription of the mPomt2 gene is initiated immediately upstream of exon 1. Northern blot and 5'-RACE showed that in testis tissue these transcripts represent only a rare population of mRNA species. Interestingly, due to differential transcription initiation in testis the vast majority of mPomt2 transcripts contain a 430-bp extended version of exon 1 (Figures 1 and 6B). The use of alternative tissue-specific promoters to produce mRNAs with novel 5' sequences is a commonly observed phenomenon in mammalian germ cells.
In many cases these transcript variations result in (1) modulation of binding sites for RNA-binding proteins involved in posttranscriptional control of gene expression, and/or (2) formation of extra translation start sites to produce testis specific protein isoforms (Hecht, 1998; Eddy, 1998
). Both possibilities might apply to mPomt2. First, our data show that the mPomt2 gene is expressed in spermatocytes; the mPOMT2 protein, however, is present predominantly in early spermatids, suggesting posttranscriptional regulation of mPomt2 expression. Therefore, we searched within the mPomt2 testis transcript for target motifs of RNA-binding proteins known to be involved in preventing premature translation. We identified only one sequence pattern (CA/TGAGCCG/CTGAGCC/T) that features homology to the conserved Y-element that serves as a binding site for the translational repressor testis/brain RNA-binding protein (Han et al., 1995
). This sequence pattern at position bp 560573, however, is located outside of the testis-specific 5' sequence (Figure 1). Second, analysis of the 5' sequence of the testis-specific mPomt2 transcript revealed an ATG-start codon at position bp 275, which is in frame with the mPomt2 ATG-start of exon 1 at position bp 485 (Figure 1). Consequently an extended ORF originates that codes for a deduced mPOMT2 protein isoform featuring 70 additive N-terminal amino acids. Our western data (Figure 7B) suggest that in testis translation of mPOMT2 is initiated at the ATG-start codon at position 275 (Figure 1), giving rise to a testis-specific protein isoform. Many examples circumstantiate that testis specific protein isoforms show enhanced biosynthetic activity or completely new functional features in germ cells (Hecht, 1998
). However, the actual function of the extended N-terminal region of the mPOMT2 protein isoform in testis remains to be elucidated in the future.
Our immunolocalization experiments revealed that mPOMT2 is located in two different subcellular compartments: the ER and the acrosome, which is a sperm-specific organelle essential for fertilization in vertebrates (Figures 5, 8, and 9). Because the acrosome is mostly derived from Golgi vesicles (Burgos and Fawcett, 1955; Clermont and Tang, 1985
; Fawcett and Hollenberg, 1963
; Susi et al., 1971
) the acrosomal localization of mPOMT2 was unexpected. However, an "extra-Golgi pathway" that directs proteins directly from the ER to the acrosome has been postulated (Tanii et al., 1992
). Furthermore, a direct contribution of ER cisternae to the Golgi complex and subsequently to the acrosome also cannot be ruled out, because the ER-resident protein calreticulin is targeted to the acrosome via the Golgi apparatus (Nakamura et al., 1993
). Acrosomal targeting signals are not yet identified. The elucidation of how mPOMT2 is targeted to the acrosome will be a goal of obvious interest in future studies.
What is the functional role of mPOMT2 mannosyltransferase in sperm cells? A number of different glycans and glycosyltransferases perform important functions during mammalian fertilization (Dell et al., 1999). One interesting example, ß1,4-galactosyltransferase (GalT), is thought to be involved in spermegg binding (Nixon et al., 2001
). Due to differential transcription initiation, two isoforms of GalT are expressed. A testis-specific protein is present in the sperm plasma membrane and might act as a lectin-like receptor that could be involved in spermegg binding. The N-terminal cytoplasmic domain of the testis GalT isoform is proposed to be involved in the activation of intracellular signaling pathways after spermegg contact is established (Shi et al., 2001
). In contrast, the somatic GalT isoform is normally confined to the Golgi, where it serves a purely biosynthetic function (Nixon et al., 2001
; Lu and Shur, 1997
). One could imagine a similar scenario for mPOMT2 in that the ER-localized protein might be involved in the synthesis of O-mannosyl glycans; in sperm mPOMT2 might have a more specific role, such as a lectin important for adhesive interactions of sperm and egg during fertilization. However, whether mPOMT2 in somatic and sperm cells has functionally distinct roles or, rather, has similar if not identical biosynthetic functions remains one of the major questions to be answered in the future.
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Materials and methods |
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Cell lines and culture
Wild-type 293 human kidney fibroblast cells (Graham et al., 1977) were cultured conventionally in Dulbecco modified Eagles medium/Hams F-12 medium supplemented with 10% fetal calf serum (Biochrom), 60 µg/ml penicillin, and 100 µg/ml streptomycin. Transfectants were selected and maintained in 200 µg/ml hygromycin (Roche). Cell lines were cultivated at 37°C in a 5% CO2 incubator.
Isolation of full-length cDNA and genomic POMT2 clones
IMAGE Consortium human POMT2 cDNA clone (Genbank Acc: AA496399; clone Id: IMAGE:755278) and mouse Pomt2 EST clone (Genbank Acc: AA711416; clone Id: IMAGE:1181688) were obtained from RZPD (Berlin) and sequenced using GSPs. Full-length human POMT2 cDNA clone (pCE2) was recovered by screening a cerebellum cDNA filter library (Lib. No.:577) supplied by RZPD with a [32P]dCTP-labeled 0.8-kb EcoRI fragment isolated from cDNA clone IMAGE:755278. Full-length mouse Pomt2 cDNA sequence was assembled by oligo-capping and RLM-RACE methods with the GeneRacer kit (Invitrogen). Following the instructions of the manufacturer, RT of mPomt2 cDNA was performed on total ligated RNA from mouse liver and testis (Clontech) with the GSP 711416.f2 (5'-CCTGAGAAGCGCAGGCCCTG-3') or GeneRacerTM OligodT primer, respectively. 5'-RACE was done using GeneRacer 5'Primer in combination with GSP tw194 (5'-gcgtcaccacagccaacgtggc-3'), and 3'-RACE using GeneRacer 3'Primer and GeneRacer 3'NestedPrimer in combination with GSP tw227 (5'-CTGTCTACCACTTAAGCTCAGAG-3') and tw230 (5'-TGTCACACTGAGGGCAGAAGG-3'), respectively.
A genomic DNA clone of mouse Pomt2 (mP2g3: ICRFP703O24280Q5) was obtained by screening a genomic mouse C57/Black6 P1 phage filter library (Lib. No.:703) supplied by RZPD using a [32P]dCTP-labeled 449-bp PCR fragment amplified on mPomt2 EST clone IMAGE:1181688 with the primer pair 711416.1 (5'-GTTCACATCCAAGCCCTGGC-3') and 711416.2 (5'-TCAGAAGCAGGCTCAGGATTCC-3'). The filter library was prehybridized for 30 min, hybridized overnight in 7% SDS, 0.5 M NaPO4, pH 7.2, 1 mM ethylenediamine tetra-acetic acid (EDTA) at 65°C and washed twice in 0.1% SDS, 40 mM NaPO4, pH 7.2, at 65°C for 30 min. Using clone mP2g3 as a template genomic fragments of the mPomt2 gene were amplified by PCR with exon-specific primers, subcloned, and sequenced. A full-length Drosophila POMT2 cDNA clone (Clone Id: LP01681) was purchased from Research Genetics, Huntsville/USA, and sequenced using GSPs.
Northern blot analyses
According to the manufacturers instructions mouse MTN-blots (Clontech) and a mouse RNA Master blot (Clontech) were hybridized with [32P]dCTP-labeled mPomt2 cDNA fragments amplified by PCR. The oligo pair tw98 (5'gggaagttactatattaaccgcac-3')/tw45 (5'-GAAGAACATCAGGATGGGGTC-3') was used to amplify a 331-bp cDNA fragment specifically hybridizing with exons 25. Furthermore, a 296-bp cDNA fragment was amplified with oligo pair tw190 (5'-AGCTCTTCTCCGCGTTTCTCAGG-3')/tw204 (5'-gaggcggcctgaggcgtagagc-3') specifically hybridizing with the 5'-extended exon 1 region of the testis-specific mPomt2 mRNA. As internal controls, the blot supplied control ß-actin was hybridized to the mouse MTN blot, and both ß-actin and ubiquitin were hybridized to the RNA Master blot.
Similarly, a Human Multiple-Tissue Expression Array (Clontech) was used to analyze differential expression of hPOMT2 with the 0.8-kb EcoRI fragment from IMAGE:755278, described earlier. Normalization was done with manufacturer-supplied ubiquitin and ß-actin probe.
Production of polyclonal anti-human POMT2 antibodies in rabbits
We immunized rabbits with a recombinant fusion protein consisting of GST and the amino acids valine-373 to leucine-470 of human POMT2. The DNA fragment encoding a putative ER-facing domain of human POMT2 was amplified by PCR using adapter oligonucleotides tw9 (5'-cgcGGATCCgtcaccacctatttgcacaag 3'; BamHI site is underscored) and tw8 (5'-ccgGAATTCccagcactttgatccggtttcc-3'; EcoRI site is underscored). The respective cDNA-fragment was combined with the GST sequence by EcoRI/BamHI subcloning into a pGEX-2TK expression vector (Pharmacia). The fusion protein was expressed in E. coli host BL21 and analyzed with SDSPAGE. The recombinant protein was excised from SDSpolyacrylamide gels and injected into rabbits. Immunizations were done by Pineda Antikoerper-Service (Berlin). Antibodies were affinity purified by binding to nitrocellulose derivatized with the GST fusion protein following the protocol of Olmsted (1981).
Expression of hPOMT2 in 293 human kidney fibroblast cell line
For construction of the human POMT2HA expression plasmid pTW49 the coding region was amplified by PCR with the primer pair SB232 (5'-atccCCGCGGTcagacaaagtgtgcctc-3'; SacII site is underscored)/SB218 (5'-acggcCTCGAGaaagtcccatgagtccagcc-3'; XhoI site is underscored) using clone pCE2 as template. The PCR fragment was subcloned as a SacIIXhoI fragment into a pCEP4 (Invitrogen)-based vector (SacIISalI), resulting in a C-terminal triple HA-tagged hPOMT2 fusion. Wild-type 293 fibroblasts were transfected using the calcium phosphate method according to Chen and Okayama (1987). Twenty micrograms of plasmid DNA of pCEP4 and pTW49, respectively, were used.
Isolation of crude membrane fractions
Fibroblast cells were cultured on 150-mm dishes and grown to confluency. Four plates, approximately 3 x 109 cells, were harvested, washed with 10 ml membrane buffer (50 mM TrisHCl, pH 7.5, 0.3 mM MgCl2), and resuspended in 2 ml of the same buffer containing protease inhibitors (1 mM phenylmethylsulfonyl, 1 mM benzamidine, 0.25 mM TLCK, 50 mg/ml TPCK, 10 mg/ml antipain, 1 mg/ml leupeptin, and 1 mg/ml pepstatin). The cells were lysed by approximately 20 strokes in a 5-ml glass homogenizer. After centrifugation at 3000 x g for 10 min at 4°C the supernatant was saved to isolate crude membranes by a 30-min 20,000 x g centrifugation at 4°C in a Sorvall SS34 rotor. Membranes were washed in TM buffer (50 mM TrisHCl, pH 7.5, 50 mM MgCl2) containing 500 mM NaCl, centrifuged, and resuspended in TM buffer containing 15% glycerol. A similar procedure was performed for the preparation of crude membranes from mouse tissues. First, the tissues were minced with forceps in cold phosphate buffered saline (PBS) including protease inhibitors, followed by lysis in a glass homogenizer and centrifugation as described.
Western blot analyses
Crude membrane fractions from fibroblasts (10 µg protein) or from different mouse tissues (80240 µg protein) were fractionated on 8% SDSpolyacrylamide gels and transferred to nitrocellulose. Affinity-purified polyclonal anti-POMT2 antibodies were used at a 1:1000 dilution; monoclonal anti-HA antibodies (16B12; Babco) were used at a 1:5000 dilution. Proteins were visualized by enhanced chemiluminescence using the Amersham ECL system. Improved sensitivity in visualizing proteins was achieved using SuperSignal (Pierce) and Hyperfilm MP (Amersham Pharmacia).
Immunofluorescence on human fibroblast cells
pTW49-expressing fibroblast cells cultured on 14-mm-diameter microscopic slides were washed with Tris-buffered saline (TBS), fixed with freshly prepared 4% paraformaldehyde in TBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100 in TBS for 10 min, and incubated in TBS containing 5% dry milk for 30 min to block nonspecific binding. Cells were then incubated with FITC-conjugated monoclonal mouse anti-HA antibodies (16B12, Babco; 1:1000 in TBS containing 0.1% Tween 20 and 2% dry milk) and polyclonal rabbit anti-ERp72 antibodies (Calbiochem; 1:1000 in TBS/0.1% Tween/2% dry milk) overnight at 4°C. Fibroblasts were washed three times in TBS/0.1% Tween for 10 min at room temperature and then incubated with TRITC-conjugated goat anti-rabbit secondary antibodies (Calbiochem; 1:250 in TBS/0.1% Tween/2% dry milk) for 1 h at room temperature. Cells were again washed three times in TBS/0.1% Tween for 10 min at room temperature and mounted with ProLong Antifade kit (Molecular Probes). Fibroblast cells were allowed to cure overnight and examined using a Zeiss Axioskop microscope equipped with epifluorescence.
Preparation of spermatogenic cells
A crude preparation of germ cells and testicular spermatids was obtained from decapsulated testes of a 6-month-old adult NMRI mouse. Testes were minced in TBS, and released cells were allowed to adhere to poly-L-lysine (Sigma)coated microscopic slides. The cells were fixed for 5 min in 100% methanol at 20°C. After blocking (30 min in TBS/0.1% Tween/5% dry milk), slides were incubated with affinity-purified anti-hPOMT2 antibodies (1:250). The cells were washed and incubated with secondary FITC-conjugated goat anti-rabbit antibodies (1:500) for POMT2 detection. As acrosome marker, the TRITC-conjugated lectin PNA from Arachis hypogaea (Sigma) was used (10 µg/ml). DNA was stained by a 5-min incubation with 0.2 µg/ml 4',6-diamidino-2-phenylindol in TBS. After a final wash, cells were mounted with ProLong Antifade kit (Molecular Probes) and cured overnight.
In situ hybridization and immunohistochemistry
To generate radiolabeled antisense and sense probes for mPomt2, a 331-bp fragment was amplified by PCR using the oligo pair tw98/tw45 (see northern blot analyses) and cloned into pGEM-T Easy (Promega). The resulting clones were analyzed by restriction digestion with XhoI/SalI, and antisense and sense constructs were identified. For the mPomt2 antisense and sense probe the constructs were linearized with SpeI and in vitro transcribed with T7 RNA polymerase using 35S-UTP.
For in situ hybridization adult mouse testis slides (Novagen) were used. Paraffin was removed according to the instructions of the manufacturer and slides were treated as described by Truernit et al. (1999). Tissue sections were incubated with the labeled probes overnight at 50°C in 50% formamide, 10% dextransulfate, 0.3 M NaCl, 1x Denhards solution, 45 mM dithiothreitol, 10 mM TrisHCl (pH 7.5), and 1 mM EDTA. After hybridization, sections were washed twice for 45 min at 45°C in 1x SSC (0.15 M NaCl, 0.015 M sodium citrate), 50% formamide, and 10 mM dithiothreitol. After an additional 5-min wash at room temperature in 1x SSC, 10 mM dithiothreitol, the sections were treated with RNaseA (20 mg/ml in 10 mM TrisHCl, pH 8.0) for 30 min at 37°C. The sections were washed again twice for 45 min at 45°C in 1x SSC, 50% formamide, 10 mM dithiothreitol, and once for 5 min at room temperature in 1x SSC, 10 mM dithiothreitol. Finally, the sections were dipped in Kodak NTB2 fotoemulsion (Integra, Fernwald, Germany) and exposed. Tissues were counterstained for 1 min in 0.5% (w/v) methyl green (Sigma).
For immunhistochemistry, tissue samples of adult mouse testes (NMRI strain, 12 months old) were fixed in Bouins solution or in methanol/glacial acid (ratio 2:1, v/v) for 12 h, dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Serial 5-µm sections were cut from paraffin blocks on a Leitz microtome and collected on gelatine/chrom alaun coated microscope slides.
Following deparaffinization, the presence of POMT2 was demonstrated immunohistochemically by streptavidin-biotin horseradish peroxidase complex (ABC) technique (Hsu et al., 1981). Endogenous peroxidase activity was eliminated by incubation with 0.5% (v/v) hydrogen peroxide solution in absolute methanol for 15 min at 20°C, and nonspecific protein binding was avoided by incubation with 10% normal goat serum in PBS for 1 h at 20°C. Sections were incubated with the affinity-purified anti-POMT2 antibodies (dilution 1:200) for 18 h at 4°C. Incubation for 1 h with biotinylated goat anti-rabbit IgG, 1:400 (Amersham-Pharmacia), followed. The sections were then reacted with ABC reagent from a commercial kit (Vector Laboratories). The bound complex was visualized with 0.05% 3,3'diaminobenzidine hydrochloride and 0.0006% hydrogen peroxide in 0.1 M PBS. Between each step sections were washed three times in PBS and once in PBS/1% bovine serum albumin. All incubations were carried out in humidified chambers to prevent evaporation. Sections were counterstained in Mayers hematoxylin, dehydrated, cleared, and mounted. Controls were performed by (1) preadsorption of the anti-POMT2 antibodies with the respective antigen (20 µg/ml) for 2 h; (2) replacing of the primary antibodies with nonimmune serum; (3) its substitution with buffer; and (4) incubation with diaminobenzidine reagent alone to exclude the possibility of nonsuppressed endogenous peroxidase activity. Lack of detectable staining of tissue elements in the controls demonstrated the specificity of the reactions.
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