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¹ Division of Infectious Disease Control, Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173-8610, Japan
² Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan
³ Laboratory of Neurochemistry, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan
⁴ Department of Neuromuscular Research, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan

	Abstract

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Dystroglycan (DG) is a widely expressed, transmembrane glycoprotein complex that plays important roles by connecting the extracellular matrix to the cytoskeleton. The - and ß-DG subunits are produced by the cleavage of residues 653 and 654 of the precursor. To clarify the mechanisms involved in cleavage and subunit association, we performed a series of mutation analyses and made the following discoveries: (i) Disruption of the intramolecular disulfide bridge between Cys669 and Cys713 in ß-DG completely abolishes the cleavage, (ii) deletions in the loop region (669–713) and in the C-terminal region of -DG (550–645) abolish the cleavage, (iii) disruption of the disulfide bridge and deletions in the loop region deteriorate the - and ß-DG subunit association, and (iv) at the cleavage site, especially, positions P1' (Ser654) and P6' (Trp659) are critical. Thus, the critical role of the Cys669–Cys713 disulfide bridge formation is, most likely, to form a specific tertiary structure, in which the - and ß-DG domains interact and the cleavage site becomes susceptible to proteolytic reactions. The Cys669 and Cys713 pair is broadly conserved in vertebrates and in some invertebrates, suggesting that the disulfide bridge formation was established early in the evolution of DG.

	Introduction

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Dystroglycan (DG), most famously known as a central component of dystrophin-glycoprotein complex in muscle, is a widely expressed, transmembrane glycoprotein complex which links the intracellular cytoskeleton and the extracellular matrices such as the basement membrane. DG is expressed in muscle and various nonmuscle tissues including nerve and epithelia and involved in early development (Williamson et al. 1997), morphogenesis (Durbeej et al. 1995) and the pathogenesis of muscular dystrophies (Michele & Campbell 2003; Cohn 2005). It has been known recently that defects in DG are associated with epithelial cancer progression (Muschler et al. 2002). DG is also a receptor for arenaviruses (Cao et al. 1998) and Mycobacterium leprae (Rambukkana et al. 1998). DG is expressed from a single gene, DAG1, which encodes an 895 amino acid precursor (Ibraghimov-Beskrovnaya et al. 1993). The precursor DG is post-translationally cleaved between amino acid residues 653 and 654 (Smalheiser & Kim 1995) to generate -DG (the N-terminal 653 amino acids) and ß-DG (the C-terminal 242 amino acids) subunits. -DG undergoes N-linked and extensive O-linked glycosylation in a tissue-specific manner through the endoplasmic reticulum (ER) and Golgi apparatus, located on the extracellular face of the plasma membrane. -DG is noncovalently anchored by ß-DG and functions as a receptor for extracellular matrix proteins, such as laminin (Smalheiser & Schwartz 1987) and agrin (Bowe et al. 1994). A defect in the O-linked sugar chains causes a loss of linkage between the cells and the extracellular matrices, such as the basement membrane, resulting in muscular dystrophies and brain abnormalities (Michele et al. 2002). ß-DG is a transmembrane protein consisting of the N-terminal extracellular domain, functioning as an anchor for -DG, the transmembrane domain, and the C-terminal intracellular domain associated with dystrophin and utrophin (Cohn 2005). ß-DG is also post-translationally glycosylated. The region responsible for the association of - and ß-DG is determined biochemically between residues 550 and 585 (especially 550 and 565) in -DG (Bozzi et al. 2001) and between 691 and 719 in ß-DG (Bozzi et al. 2003).

The importance of DG cleavage was inferred based on the finding that muscular dystrophy occurs in transgenic mice expressing an uncleavable DG mutant, Ser654Ala (Jayasinha et al. 2003); however, the precise mechanisms of DG cleavage are unknown as the protease responsible for DG cleavage has not yet been identified. In this study, we tried to determine which region and which amino acid residues in DG are critical for DG cleavage. We performed mutation analyses in which a human wild-type or mutant DG precursor was transfected into human embryonic kidney (HEK) 293 cells, and the cleaved products of DG were then detected by immunoblotting. We thus identified the unique mechanisms involved in DG cleavage: (i) The intramolecular disulfide bridge between Cys669 and Cys713 (Deyst et al. 1995) and the resultant loop region is critical for cleavage, and (ii) the C-terminal region of -DG (550–645) is also important for cleavage. We also found that both the disulfide bridge formation (669–713) and the resultant loop region are critical for the association of the - and ß-DG subunits. Presumably, the formation of a highly specific tertiary structure of DG based on the disulfide bridge formation allows its cleavage site to interact with a partner protease. Generally, the intramolecular disulfide bridge plays important roles in the structure and function of proteins. In the present study, we gained a new insight into the function of the intramolecular disulfide bridge, which is linked to site-directed cleavage of the protein.

	Results

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Characterization of DG precursor cleavage and abnormal cleavage pattern found in a mutant Cys669Arg DG

We transfected HEK 293 cells with human wild-type or various mutant DGs, and the cleaved products - and ß-DG were detected by immunoblotting. To detect -DG, we selected the anti--DG scFv phage antibody (Clackson et al. 1991), 3–18, because the widely used anti--DG monoclonal antibodies VIA4-1 and IIH6C4 could not detect -DG expressed in HEK 293 cells. The phage antibody 3–18 could detect 1 pg of the recombinant antigen MBP-DG (Fig. 1A) and -DGs expressed in HEK 293 cells (Fig. 1B,C). When the wild-type -DG alone was expressed in HEK 293 cells, a broad band around 90 kDa was detected by 3–18 (Fig. 1C). Next, when the wild-type full DG (precursor, DGwt) was expressed, a broad band or bands about 90–105 kDa and a strong band at 130 kDa (indicated by arrow) were detected by 3–18, suggesting that the band(s) at about 90–105 kDa represent cleaved -DG modified by post-translational glycosylation. Interestingly, among full DG clones obtained by RT-PCR, we identified a clone W2, which, compared with wild-type DG, had a slightly different pattern on the immunoblot with 3–18; strong single bands were detected at 105 kDa (asterisk) and 130 kDa (arrow). We then detected ß-DG by using a commercial anti-ß-DG antibody, C-20, which reacts with the C-terminus of ß-DG (Fig. 1C, right). For wild-type full DG, a strong band at 45 kDa, corresponding to cleaved ß-DG, and a band at 130 kDa (arrow) was detected. Surprisingly, the 45 kDa band was not detected for W2 DG by C-20; only a strong band at 130 kDa and a weaker band at 105 kDa were detected. Because the 130 kDa band was clearly detected by both anti--DG and anti-ß-DG antibodies, we concluded that the 130 kDa band represents uncleaved DG. In addition, because the band at 105 kDa was also detected by both anti-- and anti-ß-DG antibodies, although it was weak in the ß-DG blot, this band can be considered another form of uncleaved DG, probably a less-glycosylated form or a proteolytic product which lacks residues near the N-terminus or the C-terminus. Thus, no bands of normally cleaved - and ß-DG were detected in W2 DG-transfected HEK 293 cells. Therefore, we concluded that the clone W2 is an uncleavable DG mutant. The clone W2 had a single point mutation, Cys669Arg, at amino acid residue 669, suggesting that the substitution of the residue Cys669 abolishes the cleavage. Defective cleavage of the W2 DG precursor was also observed in COS7, CHO, and C2C12 cells when W2 DG was expressed in these cells (data not shown). We confirmed membrane localization of W2 DG by using laser confocal microscopy (Fig. 1D). Because HEK 293 cells were easily detached from the dish bottom during staining, we used CHO cells. We transfected CHO cells with wild-type or W2 (Cys669Arg) DG and then the cells were allowed to react with anti--DG (3–18) and anti-ß-DG (F15, which recognizes the extracellular domain) antibodies without membrane permeabilization. In cases, the wild-type and Cys669Arg, both - and ß-DG were detected on the cell surface, indicating that the uncleaved DG mutant was also localized on cell membrane as well as wild-type DG (Fig. 1D).

View larger version (21K): [in this window] [in a new window]   Figure 1  Expression and detection of - and ß-DG. (A) Detection of 1 pg recombinant -DG by scFv phage antibody 3–18. Left: Purified MBP (left lane) and MBP-DG (right lane) stained by Coomassie Brilliant Blue. Right: Dot blot analysis to confirm the sensitivity of 3–18. The indicated amount of purified MBP-DG dissolved in 0.5 µL of TBS was spotted on nitrocellulose membrane Hybond ECL (Amersham Biosciences, Piscataway, NJ, USA) and detected by using 3–18 as in immunoblotting (see Experimental procedures section). (B) Constructs of -DG and full DG (DG precursor) transfected into HEK 293 cells. The constructs have a signal sequence at the N-terminus (residues 1–29). The mucin-like region which undergoes extensive O-linked glycosylation is shadowed. TM indicates transmembrane domain present in ß-DG. (C) The - and ß-DG produced in HEK 293 cells. Each DG cDNA cloned into an expression vector was transfected into HEK 293 cells and detected by immunoblotting using an anti -DG scFv phage 3–18 (left) or an anti ß-DG antibody C-20 (Santa Cruz, Santa Cruz, CA, USA) which reacts with the C-terminus of ß-DG (right). The arrow shows a 130 kDa band corresponding to uncleaved DG and the asterisk shows a 105 kDa band corresponding to an another form of uncleaved DG. (D) Membrane localization of wild-type and Cys669Arg DG. CHO cells were transfected with wild-type (top) or W2 (Cys669Arg, bottom) DG. DG on the cell surface was stained by using anti--DG phage antibody 3–18 (left) and anti-ß-DG antibody F15 (center) which recognizes the extracellular domain. Nomarski images are in right.

Next, we tried to clarify the mechanisms of the defective cleavage found in the Cys669Arg mutant. Because position 669 is only 15 amino acid residues distant from the cleavage site (between 653 and 654), we first tried to determine whether the amino acid sequence around 669 or the residue cysteine itself is critical for the cleavage. We performed alanine scanning for residues 666–672 of DG (Fig. 2A,B). If the sequence context around 669 is critical, cleavage of some alanine mutants might be affected. Among the seven alanine mutants, only Cys669Ala (DGC669A) showed defective cleavage as well as Cys669Arg (W2) shown in Fig. 1, where neither the -DG (90–105 kDa) nor the ß-DG (45 kDa) band was detected (Fig. 2B). On the other hand, all the other six alanine mutants around residue 669 were normally cleaved to - and ß-DG. These results indicate that the cysteine of residue 669 is critical for DG cleavage, while the sequence context around the Cys669 is not critical.

View larger version (28K): [in this window] [in a new window]   Figure 2  Disufide bridge formation between Cys669 and Cys713 is critical for the cleavage of DG. (A) and (B): Effects of alanine substitution around the residue Cys669 (Leu666–Glu672) on the cleavage of DG. (A) Structure of the DG precursor and the sequence around Cys669. (B) Cleavage of each alanine mutant in HEK 293 cells. Top panel, -DG blot detected by 3–18; bottom panel, ß-DG blot detected by C-20. The arrow and asterisk indicate 130 and 105 kDa bands of uncleaved DG, respectively. (C) and (D): Effects of alanine substitution for each cysteine residue in DG on the cleavage of DG. (C) Cysteine residues present in DG. (D) Each cysteine residue shown in (C) was substituted with alanine and the cleavage was examined by immunoblotting; -DG was detected by 3–18 (top panel), and ß-DG by C-20 (bottom panel). The arrow and asterisk indicate 130 and 105 kDa bands of uncleaved DG, respectively.

The loop region of ß-DG and the C-terminal region of -DG are critical for DG cleavage

We further tried to identify amino acid residues that are related to DG cleavage. We performed a series of deletion studies in the N-terminal region of ß-DG (Fig. 3A,B). Mutant full DG with deletions in various regions was expressed in HEK 293 cells, and its cleavage was observed as described above. We produced deletions in a loop region between Cys669 and Cys713 (DGßd1–DGßd3) by disulfide bridge formation and in the region downstream of Cys713 (DGßd4), previously reported as a structurally flexible portion (Bozzi et al. 2003). As indicated by deletions in the loop region, the cleavage of DG is abolished, suggesting that the loop structure is required for the cleavage (Fig. 3B, DGßd1, DGßd2, and DGßd3). We also tested the deletions of residues 660–676 and 706–722, which eliminate Cys669 and Cys713, respectively, and found, as expected, that their cleavage was completely abolished (data not shown). In contrast, a deletion downstream of Cys713 (DGßd4, 723–742), located between the loop region and the transmembrane domain, did not affect DG cleavage (Fig. 3B), suggesting that the spatial distance between the membrane surface and the cleavage site is not a critical factor for cleavage.

View larger version (28K): [in this window] [in a new window]   Figure 3  The N-terminal region of ß-DG and the C-terminal region of -DG are important for the cleavage of DG. (A) and (B): Effects of deletions in the extracellular domain of ß-DG on the cleavage of DG. (A) Deletions produced in the extracellular domain of ß-DG region. The deleted residues are as follows: d1, 677–690; d2, 692–705; d3, 677–705; d4, 723–742. (B) Immunoblot analysis for each deletion mutant shown in (A). The top panel shows -DG blot detected by 3–18; the bottom panel shows ß-DG blot detected by C-20. The arrow and asterisk indicate 130 and 105 kDa bands of uncleaved DG, respectively. (C) and (D): Effects of deletions in the C-terminal region of -DG on the cleavage of DG. (C) Deletions produced in the C-terminal region of -DG. The deleted residues are as follows: d1, 520–534; d2, 535–549; d3, 550–565; d4, 570–585; d5, 586–600; d6, 601–615; d7, 616–630; d8, 631–645. (D) Immunoblot analysis for each deletion mutant shown in (C). The top panel shows -DG blot detected by 3–18; the bottom panel shows ß-DG blot detected by C-20. The arrow and asterisk indicate 130 and 105 kDa bands of uncleaved DG, respectively.

Disulfide bridge formation and the resultant loop region in ß-DG are also important for the association of - and ß-DG

The association of - and ß-DG is indispensable for the function of the DG complex, which links the extracellular matrix, plasma membrane, and intracellular cytoskeleton. We tried to determine whether the N-terminal region of ß-DG, especially the disulfide bridge and the resulting loop region between Cys669 and Cys713, is involved in the association of - and ß-DG (Fig. 4). For this purpose, separately constructed myc-tagged -DG (DG-myc) and non-tagged wild-type or mutant ß-DG, both with the same signal sequence at their N-terminus (Fig. 4A, residues 1–29), were co-expressed in HEK 293 cells. Then the - and ß-DG complex was immunoprecipitated with anti-myc antibodies. When - and ß-DG interact, the immunoprecipitate with anti-myc antibody should contain both - and ß-DG. Prior to this binding experiment, we confirmed that the intramolecular disulfide bridge between Cys669 and Cys713 was formed when ß-DG was expressed alone (not as a full DG precursor). The disulfide bridge formation was tested as described previously (Deyst et al. 1995) by comparing the mobility of ß-DG on SDS-PAGE between wild-type and mutant for reduced or non-reduced preparation of the sample (Fig. 4B). The wild-type ß-DG (ßDGwt in Fig. 4B) expressed in HEK 293 cells shows a small difference in mobility between the reduced and non-reduced samples (DTT+ and –, respectively), where the non-reduced ß-DG ran faster (indicated by arrowhead), indicating that an intramolecular disulfide bridge had been formed. Furthermore, a ß-DG mutant Cys669Ala, which lack the disulfide-bridge formation, do not show such a difference in mobility between the reduced and non-reduced samples (ßDGC669A, Fig. 4B). Similarly, no difference was found in mobility of Cys713Ala DG between the reduced and non-reduced samples (data not shown). These results indicate that the disulfide bridge between Cys669 and Cys713 is correctly formed when ß-DG alone is expressed in HEK 293 cells.

View larger version (23K): [in this window] [in a new window]   Figure 4  Disruption of the Cys669–Cys713 disulfide bridge and deletions within the loop region (660–713) abolish the binding of - and ß-DG. Whether the N-terminal region of ß-DG, especially the disulfide bridge and the resulting loop region between Cys669 and Cys713, is involved in the binding of - and ß-DG was determined. For this purpose, separately constructed myc-tagged -DG (DG-myc) and non-tagged wild-type or mutant ß-DG were co-expressed in HEK 293 cells and the - and ß-DG complex was immunoprecipitated with anti-myc antibodies. When - and ß-DG interact, the immunoprecipitate with anti-myc antibodies should contain both - and ß-DG. (A) Constructs of - and ß-DG used for the binding experiment. Both -DG (myc-tagged at the C-terminus, DG-myc) and ß-DG (wild-type or mutant) constructs have the same signal sequence at the N-terminus (residues 1–29). (B) Confirmation of Cys669–Cys713 disulfide bridge formation for solely expressed ß-DG. The ß-DG constructs (ßDGwt and ßDGC669A) were expressed in HEK 293 cells, and their SDS-PAGE samples, prepared with or without 20 mM dithiothreitol (DTT), were analyzed on SDS-PAGE (15% gel) as described previously (Deyst et al. 1995). ß-DG was detected by immunoblot with C-20. The arrowhead denotes the band with a slightly faster mobility (ßDGwt, non-reduced). (C) Binding of - and ß-DG. Each of the ß-DG constructs shown in (A) was co-expressed with myc-tagged -DG (+DG-myc, lanes 3–11) or expressed alone (lane 2), and the binding of - and ß-DG was analyzed by immunoprecipitation and immunoblotting (see Experimental procedures section). The top panel shows the ß-DG blot detected with C-20 for the immunoprecipitate. The second panel shows the -DG blot detected with 3–18 for the immunoprecipitate to confirm that DG-myc was precipitated by an anti-myc antibody. The third and bottom panels show ß-DG blot with C-20 and -DG blot with 3–18, respectively, for the total cell lysate to confirm the efficiency of expression.

View larger version (33K): [in this window] [in a new window]   Figure 6  Sequence alignment for human and zebra fish ß-DG and its corresponding regions in Drosophila and Caenorhabditis elegans DG homologue. The alignment was performed with CLUSTAL W, provided by EMBL-EBI <http://www.ebi.ac.uk/clustalw/> for human (Accession number L19711, residues 654 to 895), zebra fish (AAM78508, residues 628 to 866), Drosophila melanogaster (AAL66367, residues 691–914), and Caenorhabditis elegans (NP509826, residues 389–584) sequences. The asterisks show identical amino acid residues among the four sequences. Cys669 and Cys713 and the corresponding cysteine residues are boxed.

We investigated which position at the cleavage site sequence is critical for the cleavage reaction by alanine scanning around the cleavage site (Fig. 5A). The alanine mutant for positions P6 (Gln648) to P6' (Trp659) was expressed in each HEK 293 cell, and cleavage was observed. As seen, the cleavage of Ser654Ala (P1') was completely abolished; similarly, that of Trp659Ala (P6') was severely impaired. In addition, the cleavage of Ile650Ala (P4), Gly653Ala (P1), and Ile655Ala (P2') was suppressed, whereas the cleavage of other alanine mutants was not affected. Interestingly, a band at about 70 kDa is seen in the ß-DG blot for the uncleavable mutants (especially for Ser654Ala), suggesting that an ectopic proteolysis is stimulated in these mutants. These results suggest that at the cleavage site, positions P1' (Ser654) and P6' (Trp659) are critical, and P4, P1, and P2' are also important for the cleavage reaction.

View larger version (29K): [in this window] [in a new window]   Figure 5  Determination of cleavage site specificity. (A) Alanine scanning around the cleavage site. Each of the cleavage site residues (Gln648–Trp659) was substituted by alanine and the cleavage was examined by immunoblotting; -DG was detected by 3–18, and ß-DG by C-20. The arrow and asterisk indicate 130 and 105 kDa bands of uncleaved DG, respectively. (B) Comparison of cleavage site sequences in human DG and other human membrane bound glycoproteins.

We also investigated the effect of a protease inhibitor on DG cleavage. We expressed wild-type DG in the presence of E64 (1, 5, 10, and 50 µM), which can inhibit intracellular cysteine protease (and some of serine protease); however, no effect on cleavage was observed under these conditions, suggesting that E64 does not inhibit cleavage. To characterize the cleavage reaction in a cell-free system, we expressed DG precursor in an in vitro translation system (Flexi^® Rabbit Reticulocyte Lysate System, Promega, Madison, WI, USA); however, no cleavage of DG was observed with or without a microsome fraction (data not shown). Our effort to analyze the DG cleavage in a cell-free system remained unsuccessful, presumably because of a lack of unidentified factors.

The intramolecular disulfide bridge Cys669–Cys713 is a broadly conserved structure in DG

Finally, we investigated whether the Cys669–Cys713 disulfide bridge is widely conserved among species. As expected, the pair Cys669 and Cys713 (or its equivalent) is completely conserved in mammalian DGs (data not shown). Similarly, this pair of cysteine residues is well conserved in Xenopus laevis (Accession number, CAD42882), Xenopus tropicalis (NP 001016518), zebra fish (AAM78508, aligned in Fig. 6), European sea bass (AAZ76709), and chicken (XM425145, amino acid residues 78–320). These findings suggest that the intramolecular disulfide bridge is a conserved structure in ß-DG among vertebrates. In this context, the cleavage site sequence (Gly/Ser-Ile-Val-Val) is also conserved in these vertebrate DGs (data not shown). Furthermore, we compare the sequence of the DG homologue in Drosophila melanogaster (AAL66367) and Caenorhabditis elegans (C. elegans, NP509826) to the human ß-DG sequence (Fig. 6). Although these DG homologues do not have the conserved cleavage site sequence Gly-Ser, the region corresponding to human ß-DG was identified by Grisoni et al. (2002) for the C. elegans protein (residues 389–584) and by a sequence alignment program, CLUSTAL W <http://www.ebi.ac.uk/clustalw/>, for the Drosophila protein (residues 691–914). Surprisingly, the pair of cysteine residues at 669 and 713 in human is also conserved in these invertebrate DG homologues, although the internal sequence for the loop region is not highly conserved (between human and Drosophila, 24.4% identical; human and C. elegans, 17.4% identical) (Fig. 6). It is very likely that the Cys669–Cys713 disulfide bridge is a conserved structure in DG from the early stage of evolution.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
Specific structure around the cleavage site of DG based on the disulfide bridge formation

In this work, we have unveiled the unique mechanisms in the production of the DG complex. The most important mechanism is the formation of the intramolecular disulfide bridge between Cys669 and Cys 713 in ß-DG, which is critical for the cleavage of DG (Fig. 2D). In addition, the C-terminal region of -DG (550–645) and the loop region between 669 and 713 are critical for cleavage, as determined through a series of deletion studies (Fig. 3). Furthermore, the bridge and the loop region are also critical for the association of the - and ß-DG subunits (Fig. 4). It is most likely that the loop region functions as the binding epitope against -DG. In this context, Bozzi et al. (2003) also reported the binding epitope at residues 691–719 through an NMR study. The necessity of the C-terminal region of -DG (550–645) for cleavage (Fig. 3D) suggests a functional association between subunit binding and cleavage because the region also comprises the binding epitope at residues 550–585 against ß-DG (Sciandra et al. 2001). It is likely that the interaction between each epitope (550–585 and 669–713) contributes to the formation of a specific tertiary structure. Considering these facts, we concluded that a specific tertiary structure of a relatively large portion of precursor DG (residues 550–713, spanning more than 160 amino acids) is critical for the cleavage. In this region, closely associated structural events may occur in the following order: (i) Cys669–Cys713 disulfide bridge formation, (ii) binding of the - and ß-DG domains between each epitope, and (iii) the cleavage of DG. A model scheme for the cleavage of DG is shown in Fig. 7A. When DG and mucins are cleaved by the same protease, the cleavage of DG may occur in the ER as in mucins (Wang et al. 2002). If it is the case, DG precursor on the inner surface of the ER forms a specific tertiary structure co- or post-translationally (cleavable conformation) based on the disulfide bridge formation, providing the cleavage site (top). When the disufide bridge formation is abolished, as in the Cys669Arg mutant, DG cannot form the cleavable conformation; therefore, cleavage does not occur (bottom). Presumably, cleavage does not occur if the cysteine residues (669 and 713) remain in a reduced state.

View larger version (19K): [in this window] [in a new window]   Figure 7  Possible mechanisms involved in the cleavage of DG. (A) Model of the proposed cleavage mechanism. The chain of wild-type DG around the cleavage site forms a cleavable conformation to provide a cleavage site susceptible to a proteolytic reaction (top). This specific structure is based on the initial disulfide bridge formation between Cys669 and Cys713 and on the subsequent binding between the loop region (669–713) and the C-terminal region of -DG (550–585). On the other hand, when the disulfide bridge formation is impaired (for example, in a Cys669Arg mutant), the binding of - and ß-DG is also abolished; thus, the specific structure for cleavable conformation cannot be adopted, and cleavage does not occur (bottom). (B) The secondary structure prediction for the region critical for the cleavage of DG. The secondary structure was predicted by the phyre program <http://www.sbg.bio.ic.ac.uk/~phyre/> for residues 546–725 of DG. The symbols h, e, and c indicate the helix, ß strand and coil, respectively. The ß strands flanking the cleavage site (arrow) are boxed.

Development of the complex formation of DG

Two important clues lead us to an understanding of how the DG complex is formed: (i) The disulfide bridge formation (Cys669–Cys713) may appear in invertebrates during the evolution of DG (Fig. 6), and (ii) the cleavage site motif of DG is common among similar membrane-bound, heavily O-glycosylated proteins with a SEA module (Fig. 5B). The latter fact suggests that these proteins are cleaved by the same or a very closely associated protease, although the region around the cleavage site of DG is not a SEA module. Levitin et al. (2005) reported that the MUC1 SEA module is a self-cleaving domain. At present, whether the main mechanism of the cleavage of DG is an autocatalytic reaction is unknown. Recently, the solution structure of the SEA module was clarified for the MUC16 cancer antigen (Maeda et al. 2004). The SEA module forms a unique /ß sandwich fold composed of two helices and four antiparallel ß strands and has a characteristic TY-turn. The cleavage site sequence Gly/Ser-Ile-Val-Val and similar sites are located between the ß2 and ß3 strands and presumably protrude from the entire domain as loop structures (Maeda et al. 2004; Palmai-Pallag et al. 2005). Interestingly, when the secondary structure was predicted by the phyre program <http://www.sbg.bio.ic.ac.uk/~phyre/> for residues 546–725 of DG, the cleavage site was located between two ß strands (Fig. 7B). It is likely that when DG forms the cleavable conformation, the cleavage site protrudes into a solvent as in SEA modules. It should be pointed out, however, that the dependence on the disulfide bridge formation is specific to the cleavage mechanism of DG but not to that of the SEA module since many of the SEA module sequences contain zero or one cysteine residue downstream of the cleavage site (Maeda et al. 2004; Palmai-Pallag et al. 2005). Because the cleavage site motif is not found in invertebrate DGs (at least in fly and nematode), the cleavage mechanisms may be gained on the way to vertebrates during evolution. These facts suggest that (i) the tertiary structure based on the disulfide bridge formation has been established as a specific structural feature of DG in an early phase of evolution, (ii) some residues in DG protrude into a solvent, and (iii) by gaining a cleavage site sequence by, for example, mutational changes in the protruded residues, DG obtains the specific cleavage mechanisms of the formation of a specific tertiary structure. We do not know precisely why DG must be cleaved to produce the DG complex in vertebrates. An uncleavable DG, Ser654Ala, expressed in transgenic mice shows a dominant negative effect, resulting in muscular dystrophy, where an altered glycosylation of -DG is observed (Jayasinha et al. 2003). This suggests that the cleavage is important for faithful glycosylation of DG. However, laminin 2 is not eliminated in the muscle basement membrane of the mice (Jayasinha et al. 2003), suggesting that the binding with laminin, which is an important function of -DG, does not deteriorate in the uncleavable DG mutant. For MUC1, the mutagenesis of a Gly-Ser cleavage site inhibits ectodomain shedding mediated by secondary proteolysis (Lillehoj et al. 2003). Recently, a proteolytic processing of -DG was also reported (Kanagawa et al. 2004). It is likely that there are still unknown factors in the cleavage and the subsequent complex formation of DG.

	Experimental procedures

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Anti--DG scFv phage antibody

An anti--DG single-chain Fv (scFv) phage antibody (Clackson et al. 1991) 3–18 was selected from the mouse scFv phage display library (Tanaka et al. 2005) by using maltose binding protein-fused mouse -DG (amino acid residues 1–278, MBP-DG) as an antigen. For the construction of MBP-DG, the -DG fragment (1–278) was amplified by PCR using the restriction-site-tagged primers, 5'-TATGAATTCCACTGGCCCAGTGAACCCT-3' (with EcoRI site as underlined) and 5'-TATGTCGACTAGGGGAGAGTGGGCTTCTTA-3' (with SalI site as underlined), and mouse DAG1 gene cDNA as a template. The plasmid for the expression of MBP-DG was created by ligating the EcoRI-SalI-digested fragment of -DG into EcoRI-SalI-digested pMALc. MBP-DG was expressed in Escherichia coli TG1 and purified with amylose resin as described previously (Tanaka et al. 2000). An anti--DG scFv phage (a parent clone) was selected by three rounds of conventional panning (Griffiths et al. 1994), where scFv phages were preincubated with purified MBP (100 µg/mL) before incubation with an antigen to eliminate anti-MBP clones. The antigen affinity of the parent clone was further improved by VH-CDR3 randomization (Nielsen & Marks 2001), and finally, clone 3–18 was obtained. The 3–18 detected human -DG as well as mouse -DG.

Construction of wild-type and mutant DGs for expression

Human DG cDNA was obtained by RT-PCR from human skeletal muscle RNA (BD Biosciences Clontech, San Jose, CA, USA) with random primers (Invitrogen, USA) for reverse transcription and with forward (5'-ATCGCGGCCGCCATGAGGATGTCTGTGGGCCTC-3') and reverse (5'-GATAAGCTTGCCCCGGGTGATATTCTGCAG-3') primers for the subsequent PCR. The wild-type DG construct (DGwt) was produced by cloning of NotI-EcoRI-digested DG cDNA fragment into pcDNA3.1(-)Zeo at NotI-EcoRI sites. A variety of mutant DG constructs carrying nucleotide substitutions or deletions were created by using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocol, with primer sets designed for each DG mutant. The sequences of the primer sets are shown in Supplementary material. A construct DG (for the expression of -DG alone) was produced by ligating a NotI-HindIII-digested -DG cDNA fragment, which was amplified by using primers 5'-ATCGCGGCCGCCATGAGGATGTCTGTGGGCCTC-3' and 5'-GATAAGCTTTTAGCCCCGGGTGATATTCTGCAG-3' (with terminal codon) from DG cDNA, into the NotI-HindIII sites of pcDNA3.1-myc-His(-)A. Similarly, a construct DG-myc was produced by ligating a NotI-HindIII-digested -DG cDNA fragment, which was obtained as described above by using primers 5'-ATCGCGGCCGCCATGAGGATGTCTGTGGGCCTC-3' and 5'-GATAAGCTTGCCCCGGGTGATATTCTGCAG-3' (without terminal codon) into NotI-HindIII sites of pcDNA3.1-myc-His(-)A.

Cells and transfection

HEK 293 cells obtained from Riken BRC (JAPAN) were cultured in D-MEM supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 units/mL), and streptomycin (100 µg/mL). The cells were transfected with expression constructs for DG with the aid of Lipofectamine (Invitrogen) together with a PLUS reagent (Invitrogen) according to the manufacturer's protocol. After 60 h of transfection, the cells were collected and subjected to immunoblotting or immunoprecipitation experiments. For all expression experiments, the vector alone (pcDNA3.1(-)Zeo, without DG sequences) was transfected as negative control (Control in Figs 1–5). When necessary, COS7, CHO, and C2C12 cells were cultured in the same medium.

Immunoblotting

Proteins (15 µg for each lane) were resolved on SDS-PAGE (7.5% gel for -DG and 10% gel for ß-DG) and transferred to nitrocellulose membranes (Hybond ECL, Amersham Bioscience). The blotted membranes were preincubated with TBST (20 mM Tris–Cl, pH 7.4, 150 mM NaCl, and 0.1% v/v Tween-20) for 1 h to enhance detection sensitivity (Tanaka et al. 2002) and subsequently blocked with 5% w/v skim milk in TBS (20 mM Tris–Cl, pH 7.4 and 150 mM NaCl) for 1 h. For -DG blot, the membrane was incubated in 5% skim milk-TBS with anti--DG scFv phage 3–18 (5 x 10¹⁰ cfu/mL) for 1 h, washed with TBST 5 times for 5 min, and subsequently incubated in TBST with 10 000-fold-diluted HRP-conjugated anti-M13 antibody (Amersham Bioscience) for 30 min (Tanaka et al. 2002). For ß-DG blot, the blocked membrane was incubated with 1000-fold-diluted anti ß-DG goat polyclonal antibody C-20 (Santa Cruz Biotechnology) in 5% skim milk-TBST for 1 h, washed with TBST 5 times for 5 min, and subsequently incubated with 1000-fold-diluted HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology) in 5% skim milk-TBST for 30 min. For both blots, after being washed with TBST as above, the blot was visualized by an ECL Plus immunoblot detection system (Amersham Bioscience).

Immunofluorescence

CHO cells cultured in glass base dishes (35 mm diameter; IWAKI, Japan) were transfected with 2 µg of plasmid (pcDNA3.1(-)Zeo for control, DGwt, or W2) with the aid of Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. In order to detect DGs on the cell surface, rinsing and incubations were performed at 4 °C until fixation to suppress endocytosis. After 24 h of transfection, the cells were rinsed (4 times with PBS), blocked with 1% bovine serum albumin (BSA)-PBS for 1 h, and incubated with the mixture of primary antibodies in 1% BSA-PBS for 1 h; anti -DG phage antibody 3–18 (5x10¹⁰ cfu/mL) and anti-ß-DG goat polyclonal antibody F15 (Santa Cruz Biotechnology) which recognizes the extracellular domain at 1:50 dilution. Then the cells were rinsed and fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 15 min. After fixation, the cells were rinsed and incubated with the mixture of secondary antibodies, FITC-conjugated anti-M13 (PROGEN Biotechnik, Heidelberg, Germany) at 1:400 dilution and Alexa Fluor 555 anti-goat IgG (Molecular Probes, USA) at 1:250 dilution, in 1% BSA-PBS for 30 min at room temperature. After the cells were rinsed, the fluorescent images were collected by confocal laser scanning microscope FV1000 (Olympus Optical, Tokyo, Japan) in a sequential scanning mode. No positive signal was observed in control cells transfected with pcDNA3.1(-)Zeo without DG sequences.

Immunoprecipitation

HEK 293 cells transfected with various expression constructs for - and ß-DG were suspended with lysis buffer (20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1 mM Na₃VO₄, 1 mM NaF, 2 mM EDTA, 1% v/v Nonidet P-40) for 30 min at 4 °C and were microcentrifuged for 10 min at 15 000 rpm. The supernatant (cell lysate) was incubated overnight at 4 °C with protein G-Sepharose beads (Amersham Bioscience) and anti-c-myc monoclonal antibody 9E10 (Ab-1, Calbiochem, San Diego, CA, USA). After incubation, the beads were washed 3 times with lysis buffer, resuspended, and boiled in SDS sample buffer (50 mM Tris–Cl, pH 6.8, 2% SDS, 10% glycerol, 6% ß-mercaptoethanol). Samples were resolved by SDS-PAGE and subjected to immunoblotting.

	Acknowledgements

 
We acknowledge Drs S. Imajoh-Ohmi (The Institute of Medical Science, The University of Tokyo), E. Ozawa and M. Imamura (NCNP), and K. Kuroda (Nihon University) for suggestions and advice. We also thank Dr K. Shimizu (Nihon University) for encouragement. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Open Research Center for Genome and Infectious Disease Control (Nihon University).

	Footnotes

 
Communicated by: Yo-ichi Nabeshima

^aPresent address: Department of Bacteriology, Iwate Medical University, 19-1 Uchimaru, Morioka-shi, Iwate 020-8505, Japan

^* Correspondence: E-mail: tanakat{at}med.nihon-u.ac.jp

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Received: 24 March 2006
Accepted: 10 October 2006

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