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Published as doi: 10.1096/fj.07-8354com.
 (The FASEB Journal. 2008;22:612-621.)
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(The FASEB Journal. 2008;22:612-621.)
© 2008 FASEB

SEA domain proteolysis determines the functional composition of dystroglycan

Armin Akhavan*, Silvia N. Crivelli{dagger}, Manisha Singh*, Vishwanath R. Lingappa* and John L. Muschler*,1

* California Pacific Medical Center Research Institute, San Francisco, California, USA; and

{dagger} Lawrence Berkeley National Laboratory, Berkeley, California, USA

1Correspondence: California Pacific Medical Center Research Institute, 475 Brannan St., Ste. 220, San Francisco, CA 94107, USA. E-mail: muschler{at}cpmcri.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Post-translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand-binding and transmembrane domains into the noncovalently attached {alpha}- and β-subunits. We investigate here the structural determinants and functional significance of this cleavage. We show that cleavage of DG elicits a conspicuous change in its ligand-binding activity. Mutations that obstruct this cleavage result in increased capacity to bind laminin, in part, due to enhanced glycosylation of {alpha}-DG. Reconstitution of DG cleavage in a cell-free expression system demonstrates that cleavage takes place in the endoplasmic reticulum, providing a suitable regulatory point for later processing events. Sequence and mutational analyses reveal that the cleavage occurs within a full SEA (sea urchin, enterokinase, agrin) module with traits matching those ascribed to autoproteolysis. Thus, cleavage of DG constitutes a control point for the modulation of its ligand-binding properties, with therapeutic implications for muscular dystrophies. We provide a structural model for the cleavage domain that is validated by experimental analysis and discuss this cleavage in the context of mucin protein and SEA domain evolution. Akhavan, A., Crivelli, S. N., Singh, M., Lingappa, V. R., Muschler, J. L. SEA domain proteolysis determines the functional composition of dystroglycan.


Key Words: mucin • laminin • autoproteolysis • muscular dystrophy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DYSTROGLYCAN (DG) IS A WIDELY expressed extracellular matrix receptor with known roles in muscle, neuronal, and epithelial cell biology (1 , 2) . Genetic ablation experiments have demonstrated the involvement of DG in many cell biological processes, including laminin and basement membrane assembly, sarcolemmal stability, cell survival, cell migration, and epithelial polarization and function (2 3 4 5 6 7 8) . Importantly, alterations in DG function are linked to human diseases, including muscular dystrophies and cancers (1) .

DG is encoded by a single gene, the product of which is constitutively cleaved to form the {alpha}- and β-DG subunits. The ligand-binding {alpha}-DG subunit is composed of two globular regions separated by a mucin domain and is retained at the cell surface by noncovalent interactions with the transmembrane β-DG subunit (shown in Fig. 1 A, B). In addition to the constitutive cleavage event, the functional form of DG on the cell surface is generated through O-linked glycosylation within the mucin domain. The O-linked glycosylation of DG is critical for its ligand-binding properties, permitting binding to extracellular matrix molecules, including laminins, perlecan, and agrin (1 , 9) . Altered glycosylation of DG that reduces its ligand-binding properties has been directly linked to congenital muscular dystrophies (10) and may impact cancer progression (11) . Therefore, the factors modulating the functional composition of DG represent potential targets for therapeutic intervention (1) .


Figure 1
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  		Figure 1. The S654A mutation blocks DG precursor cleavage and results in enhanced glycosylation and laminin binding. A) Diagram of the DG heterodimer spanning the plasma membrane (PM). B) Diagram of the structural elements of DG, including the precursor cleavage site at the consensus GSIVV sequence (arrow) that divides the {alpha}- and β-DG subunits. Regions with defined structures are labeled and include an immunoglobulin-like domain (Ig-like), RNA-binding protein-like domain (RBP-like), mucin domain, and cadherin-like domain. The structure of the cleaved region between the cadherin-like domain and transmembrane (TM) domain is undefined. C) Immunoblot for β-DG in extracts from cells expressing the wild-type (WT) and mutant (S654A) DG or vector control (Vector). D) Immunoblot of these same extracts using a polyclonal anti-{alpha}-DG antiserum, or using the carbohydrate-sensitive anti-{alpha}-DG monoclonal antibody clone IIH6 (E). F) Assay of DG binding to laminin by laminin overlay. G) Detection of laminin binding and assembly at the surface of cells expressing DG mutants and controls. Phase images are paired with fluorescent imaging of FITC-laminin-111 assembly for vector, wild-type and S654A-expressing cells. (Scale bar=50 µm)

Despite the importance of post-translational modifications of DG in physiology and disease, the functional significance and the structural determinants of DG cleavage have not been resolved (12 13 14 15) . In this study, we found, unexpectedly, that blocking the DG precursor cleavage enhanced the ligand-binding capacity of the {alpha}-DG subunit. We determined that the cleavage domain of DG comprises an "SEA" (sea urchin, enterokinase, agrin) module, and we provide structural predictions for this distinct region. Our results point to cleavage of DG as an important control point for the modulation of its function, which might be exploited for treatment of muscular dystrophies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Commercially available antibodies used in this study included the monoclonal anti-C-terminal β-DG clone NCL-b-DG (Novacastra Laboratories, Newcastle on Tyne, UK), anti-E-cadherin clone 36 (BD Transduction Laboratories, San Jose, CA, USA) and anti-{alpha}-DG clone IIH6C4 (16) (Upstate, Charlottesville, VA, USA). The polyclonal anti-mouse-laminin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The polyclonal anti-{alpha}-DG antiserum was a gift from Dr. Kevin Campbell (Iowa City, IA, USA). The MANDAG2 antibody, developed by Glenn E. Morris, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences. Horseradish peroxidase-conjugated secondary antibodies specific for mouse IgG (Amersham Pharmacia, Piscataway, NJ, USA), mouse IgM (Sigma-Aldrich), and rabbit IgG (Caltag, Burlingame, CA, USA) were used for immunoblots and the laminin overlays.

Immunoblotting
Cell extracts and invertebrate protein extracts were prepared in cell lysis buffer [50 mM Tris (pH 7.4), 1% (wt/vol) sodium deoxycholate, 1% (vol/vol) Nonidet P-40, 0.1% (wt/vol) SDS, 150 mM NaCl], and protease inhibitor cocktail (Calbiochem, La Jolla, CA, USA). Insoluble material was removed by centrifugation at 12,000 g for 15 min. SDS-PAGE was performed under reducing conditions using equal amounts of protein and 4–12% or 4–20% NuPage gels (Invitrogen, Carlsbad, CA, USA). Proteins were electrophoretically transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). Blots were blocked in 5% nonfat dry milk in TBS-T (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween-20) for 1 h at RT, followed by incubation in blocking buffer overnight at 4°C with primary antibodies, then 1 h at RT with HRP-conjugated secondary antibodies diluted in blocking buffer. Blots were washed in TBS-T after antibody incubations, and bands were visualized with Supersignal chemiluminescence substrate (Pierce Biotechnology, Rockford, IL, USA). Laminin overlays were performed by electrophoretic transfer of proteins to Immobilon-P membranes as described above, followed by incubation with purified laminin-111 (Sigma-Aldrich) at 5 µg/ml in blocking buffer plus 10 µM CaCl2 for 1 h. All subsequent washes and antibody incubations included 10 µM CaCl2. The bound laminin was subsequently detected using an antilaminin polyclonal antibody (Sigma-Aldrich) followed by the horseradish peroxidase-conjugated anti-rabbit secondary antibody. Antibody binding was visualized by Supersignal chemiluminescence substrate (Pierce Biotechnology).

Cell culture, infection, and DG mutagenesis
The creation of the MEpG cell line was described previously (2) . DG cDNAs were expressed in cultured mammary epithelial cells by retroviral gene transfer using the retroviral vector pBMN-IRES-PURO, as described previously (2) . DG mutants were constructed using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and verified by sequencing. Laminin assembly in living cells was preformed as described previously (2) using laminin-111 conjugated to FITC. Fluorescent images were obtained with a Nikon Eclipse TE2000-U inverted microscope, Photometrics CoolSNAP HQ camera, MetaMorph 6.1r1 software (Universal Imaging Corporation, Downingtown, PA, USA).

Cell-free synthesis and proteolysis
DG variants were cloned into pTNT vector (Promega, Madison, WI, USA) and transcribed and translated as described previously using rabbit reticulocyte lysate in the presence or absence of canine pancreatic rough microsomal membrane (17) . Transcription and translation were performed for 90 min at 40°C and 34°C, respectively. Proteolysis was carried out at the end of translation with 0.25 µg/µl Proteinase K (PK) for 1 h on ice. PK activity was stopped with 0.2 M PMSF followed by boiling in 0.1 M Tris and 1% SDS. Protein samples were separated on 10% Tris-glycine SDS-PAGE.

Structure modeling
We performed two types of modeling: template-free and template-based. Template-based modeling requires a homologous structure to model the target protein, whereas template-free modeling relies more on physical principles. The template-free modeling used four methods considered to be among the best according to the recent CASP6 and CASP7 (6th and 7th Critical Assessment of Techniques for Protein Structure Prediction): Rosetta (18) , I-TASSER (19) , SAM-T06 (20) , and RAPTOR (21) . Template-based modeling included the following steps. First, the primary amino acid sequence of the SEA domain was submitted to secondary structure prediction servers, including JPRED, PSIPRED, SAM-T06, and PHD (22 23 24 25) . Next, ProteinShop (26) was used to create an extended conformation with secondary structure matching those predictions. Finally, we modeled the resulting structure according to the native structure of the cleaved Muc1 SEA domain (PDB number 2ACM). The distribution of the predicted secondary structure elements for DG differed from that of the Muc1 SEA domain elements; therefore, we packed the elements of secondary structure to preserve the topology of the Muc1 SEA domain, as well as to reproduce our observations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Blocking cleavage of the DG precursor results in enhanced laminin-binding capacity
Cleavage of the DG precursor is blocked by the mutation of serine-654 to alanine (S654A) at the sequence GSIVV (14 , 15) , where cleavage occurs between the glycine and serine. To asses the significance of DG cleavage, the processing and function of this cleavage-deficient DG mutant were assayed in mammary epithelial cells where DG has a clear role in the binding and assembly of laminin at the cell surface (2) . The wild-type DG and cleavage-defective mutant were expressed by retroviral gene transfer in previously generated DG–/– mammary epithelial cell lines (2) , which allow for assaying of the structure-function relationships in DG without interference from an endogenous DG.

Immunoblots of extracts from cells expressing the wild-type DG showed production of the 43 kDa β-DG, reflecting cleavage of the DG precursor into the {alpha} and β subunits (Fig. 1C ). In extracts of cells expressing the S654A mutant, the anti-β-DG antibody detected a broad ~150-kDa band, indicating the obstruction of the precursor cleavage and generation of an {alpha}/β-DG monomer (Fig. 1C ). Immunoblots using a polyclonal anti-{alpha}-DG antiserum detected the broad band of the glycosylated {alpha}-DG subunit at ~120 kDa in cells expressing the wild-type DG and the expected higher mass DG monomer in cells expressing the S654A mutant (Fig. 1D ).

A previous study indicated that the functional glycosylation of DG was perturbed by the S654A mutant (14) ; therefore, we tested the functional glycosylation of the mutant in our cells using the IIH6 monoclonal antibody and laminin-binding assays. The IIH6 monoclonal antibody binds to {alpha}-DG in a carbohydrate-dependent manner; chemical deglycosylation, or natural hypoglycosylation of {alpha}-DG eliminates the IIH6 antibody binding epitope (1 , 11 , 27 , 28) , and this epitope is enhanced by the activity of the glycsoyltransfrerase LARGE, acting on modifications of the {alpha}-DG mucin domain (9 , 27) . The antibody also competes with laminin binding to DG and is deemed to detect the functional glycosylation of the {alpha}-DG mucin domain (1 , 28) . Surprisingly, in cells expressing the S654A mutation, the higher mass DG monomer was detected much more strongly by the IIH6 antibody than the wild-type {alpha}-DG subunit (Fig. 1E ). When normalized to the levels of the core peptide, as measured by the polyclonal anti-{alpha}-DG antiserum (Fig. 1D ), the binding of the IIH6 antibody was 49% ± 10% stronger for the S654A mutant than the wild-type protein, indicating more efficient glycosylation of the DG monomer. These same results were subsequently observed when testing similar cleavage-blocking mutants, including the S654G mutation (see below).

An increased recognition by the IIH6 antibody is predicted to correspond to an increase in laminin binding by the monomeric DG. To test this, the ability of the S654A mutant to mediate laminin binding was assayed by laminin overlay. This assay revealed an increase in laminin binding by the S654A mutant, relative to the wild-type protein, that was proportional to the increased detection by the IIH6 antibody (Fig. 1F ). To establish whether this monomeric DG was functional at the cell surface, we assayed its ability to mediate laminin binding and assembly in living cells. In these assays, adherent cells were incubated with fluorescein-labeled laminin-111, which assembles on the surface of DG-expressing mammary epithelial cells but not DG–/– cells (2) . Like the wild-type DG, the S654A mutant restored the ability of DG–/– cells to bind and assemble laminin on the cell surface (Fig. 1G ). Therefore, cleavage-defective mutants can be functional laminin receptors at the cell surface.

DG cleavage is an early processing event
Given that cleavage of the DG precursor modulates its functional glycosylation, we suspected that the cleavage precedes O-linked glycosylation, possibly before transit to the Golgi complex. To test this hypothesis, we attempted to reconstitute this cleavage in a controlled cell-free protein translation system using reticulocyte lysates in the absence or presence of microsomal membranes derived from the endoplasmic reticulum (ER). In vitro synthesis of either the wild-type or S654G mutant DG, in the absence of membranes, generated one prominent band corresponding to the molecular mass of the unprocessed monomer (Fig. 2 A, indicated by upward arrows in lanes 1 and 5). When ER membranes were added, a higher mass species was generated for both the wild-type and mutant DG (indicated by * in lanes 2 and 6) which was blocked by the addition of a tripeptide inhibitor of N-linked glycosylation (AP). Therefore, the wild-type and the mutant DG proteins were translated in the cell-free system and translocated into the ER membrane, as evident by the presence of N-linked glycosylation. Importantly, in the presence of membranes, translation of the wild-type DG generates an additional species of ~80 kDa, corresponding in size to the {alpha}-DG peptide after precursor cleavage and before extensive O-linked glycosylation (downward arrow in lane 2). As expected, the {alpha}-DG product was absent from translations of the cleavage-defective mutant (lane 6).


Figure 2
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  		Figure 2. In vitro reconstitution of DG cleavage. A) The cDNA encoding WT and S654G DG genes were transcribed using SP6 polymerase and translated with reticulocyte lysate in the absence (–) or presence (+) of microsomal membrane (ER), acceptor peptide (AP), and proteinase K (PK). Protein samples were separated on SDS-PAGE and detected through radiolabeled methionine incorporated during de novo synthesis. The positions of precursor monomer ({uparrow}), N-linked glycosylated monomer (*), and {alpha}-DG ({downarrow}) are indicated. B) Schematic diagram depicting WT and S654G DG on translocation across the ER membrane before (left) and after (right) proteolysis with PK. Letters correspond to fragments shown in A, as described in the text.

To confirm that the products detected in the cell-free system exhibit the characteristics expected of the DG molecule translocated into the ER, we tested their accessibility to proteinase K (PK). As depicted in Fig. 2B , exogenous PK is expected to degrade all peptides exposed on the outside of the microsomes and to generate fragments labeled as A ({alpha}-DG and the ectodomain of β-DG, which is generated from degradation of the C-terminal tail of the monomeric DG), B ({alpha}-DG, which is expected to be fully protected from PK degradation) and C (the ectodomain of β-DG, generated by degradation of the β-DG C-terminal tail). As seen in Fig. 2A , PK treatment of wild-type DG produced the expected bands labeled A and B in lane 4. The mutant DG, however, generates a single monomer, which, on PK treatment, gives rise only to fragment A (Fig. 2A , lane 8). Collectively, the data gathered in cell-free system demonstrate that cleavage of DG takes place early during synthesis on translocation across the ER membrane.

The cleavage site that generates the {alpha}- and β-DG subunits is positioned within an SEA module
When identified in other molecules, the GSIVV cleavage site has been found to reside within an SEA domain, a distinct ~120 amino acid protein module, defined primarily by a characteristic protein folding pattern and most frequently associated with proteins displaying abundant O-linked glycosylation (29) . SEA modules display a four-stranded antiparallel β sheet (β1-β4) backed by {alpha} helices ({alpha}1-{alpha}4), as observed in the three-dimensional NMR structures of the cleaved Muc1 SEA domain (30) and the noncleaved Muc16 SEA domain (31) . The secondary structure components of the cleaved Muc1 SEA domain appear in the order β1-{alpha}1-{alpha}2-β2-β3-{alpha}3-{alpha}4-β4, and the cleavage occurs at a bend between the β2 and β3 sheets (Fig. 3 A, diagram above sequences). Figure 3A shows a sequence comparison between the SEA domains of four related human mucins (Muc1, 3, 12, and 17), all cleaved at the glycine-serine bond in the conserved GSVVV or GSIVV site, and their alignment with the primary sequence of the human DG protein. The sequence of DG that aligns with the mucins corresponds closely to the undefined region of DG shown in Fig. 1B . As seen in this comparison, the mucin SEA modules show little conservation of sequence identity, even among the most closely related molecules, such as Muc12 and 17 (32) . Similarly, alignment with DG shows very little sequence identity with the mucins, except for conservation of the consensus cleavage site. Nevertheless, methods of secondary and tertiary structure prediction devise structures for DG that reflect the consensus SEA module. Secondary structure analysis predicts all of the basic elements of a SEA domain within the DG sequence: the four β sheets, two of which immediately flank the DG cleavage site and the four {alpha} helices flanking the two central β sheets (Fig. 3A , below the sequences). The positions of the putative secondary structure elements in DG are shifted only slightly from the positions of the corresponding elements in the Muc1 SEA domain (Fig. 3A , top).


Figure 3
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  		Figure 3. Alignment of mucin SEA domain sequences with DG sequences, and structural predictions for the corresponding DG domain. A) Alignment of human mucins and DG, all sharing the GS(I/V)VV consensus cleavage site (highlighted by black arrows). Residues are color coded according to conservation (red, completely conserved residues; cyan, identical residues; yellow, similar residues). Diagrams above the alignment correspond to the known secondary structure elements of the Muc1 SEA domain (Blue arrows, β-sheets; red cylinders, {alpha}-helices). The DG amino acid sequence is repeated below this alignment along with the secondary structure predictions of the PSIPRED and SAM-T06 algorithms, showing predicted {alpha}-helical (H, red), β-sheet (E, blue), and random coil (C) structures. The four β-sheets and {alpha}-helices of the putative DG SEA domain are underlined. The amino acid numbering appears above the DG sequence. B) Predicted three-dimensional structure of the DG domain using the SAM-T06 algorithm, based only on the DG primary sequence. C) Known three-dimensional structure of the Muc1 SEA domain and cleavage site. D) Best fit model of the DG SEA domain, including the cleavage site and putative disulfide linkage between cysteines 669 and 713.

Multiple independent methods of three-dimensional structure prediction produced model structures with antiparallel β sheets, backed by {alpha}-helices (Figs. 3B and Supplemental Fig. S1). Many of these models resembled the known Muc1 structure, shown in Fig. 3C , even though the modeling programs did not use this structure as a template. Most significantly, each model of the DG structure predicted a tight turn at the junction of two β sheets surrounding the DG cleavage site (Figs. 3B and Supplemental Fig. S1). Precisely such a turn between β sheets exists at the Muc1 cleavage site (Fig. 3C ) and is deemed necessary for Muc1 cleavage (30) . By combining sequence alignments with secondary and tertiary structure predictions, we present here a "best fit" structural model for this cleaved domain of DG, shown in Fig. 3D . Highest confidence is given to the positions of the 4 β-sheets in this model, whereas the positions of the {alpha}-helices, particularly {alpha}3 and {alpha}4, are less certain. Aptly, two cysteines (C669 and C713) in the DG SEA domain sequence are positioned, in our model, immediately adjacent to each other following the β4 sheet (Fig. 3D ). These cysteines are presumed to form a disulfide bond, as indicated in our model.

The cleavage of DG exhibits defining characteristics of SEA domain autoproteolysis
The predicted existence of an SEA module in DG, and the conservation of amino acids surrounding the cleavage sites both of DG and Muc1, suggests a mechanism of cleavage that is conserved between these two molecules. For Muc1, cleavage has been determined to occur by domain-autonomous autoproteolysis (30 , 33) . Muc1 autoproteolysis appears to depend on a conformational strain at the turn between the β2 and β3 sheets and on the presence of a hydroxyl group in the serine located at the cleavage site, which is believed to play a direct role in the catalytic mechanism (30 , 33) . Consequently, mutation of hydroxyl bearing serine residue abolishes cleavage, but not when mutated to either a threonine or cysteine (33) . Also, introduction of a "G-loop" (4 glycines) immediately before the cleavage site abolishes cleavage, theoretically by relieving strain on the glycine-serine peptide bond (30) .

To test whether these known determinants of SEA autoproteolysis were critical for DG cleavage, site-specific mutations were introduced into the DG cDNA, and the mutants were expressed in the DG–/– mammary epithelial cell line. Immunoblotting for β-DG showed that mutation of Ser-654 residue to either an alanine or glycine completely abolished cleavage of the DG subunit, as shown by conversion of the 43 kDa β-DG subunit into the 150 kDa DG monomer (Fig. 4 A). On the other hand, mutation of this residue to either threonine or cysteine allowed cleavage to occur, albeit less efficiently than observed for the wild-type protein, resulting in the presence of both cleaved and monomeric DG in the same cell population (Fig. 4A ). Addition of a G-loop adjacent to the cleavage site also abolished cleavage. Mutation of the distal phenylalanine 608, which maps within the β1 sheet (Fig. 3A ), partially inhibited the cleavage of the DG precursor (Fig. 4A ), and also resulted in much lower expression levels of the protein, as evidenced by weaker detection by the β-DG antibody.


Figure 4
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  		Figure 4. Assays of DG cleavage and processing in cells expressing DG point mutations. A) Immunoblot for β-DG in extracts from cells expressing the vector control (Vector), the wild-type DG (WT), and DG point mutations. B) Immunoblot for {alpha}-DG in these same extracts using the carbohydrate-sensitive IIH6 anti-{alpha}-DG monoclonal antibody. As a control for protein loading, the same membrane was stripped and reprobed for E-cadherin.

Immunoblotting for {alpha}-DG using the IIH6 antibody showed an enhanced recognition of the monomeric DG by this antibody (Fig. 4B ), as observed previously for the S654A mutation (Fig. 1) , reflecting enhanced glycosylation. Enhanced glycosylation of the monomer was also evident within the cell populations of the partially cleaved S654T and S654C mutants, where the monomeric DG band was detected by the IIH6 antibody much more strongly than the {alpha}-DG subunit (Fig. 4B ), even though the immunoblot for β-DG in Fig. 4A indicated that approximately half of the DG present in these cell populations was cleaved. The enhanced recognition by the IIH6 antibody in Fig. 4B corresponded to increased laminin-binding capacity in all mutations, and all mutants restored the ability of DG–/– cells to bind and assemble laminin on the cell surface (data not shown).

The SEA module is present in all DG homologs
DG is an ancient molecule, with homologs identified in fruit flies (Drosophila melanogaster) and nematodes (Caenorhabditis elegans). The cleavage of DG is always observed in vertebrates, but in fruit flies and nematodes, DG is evident as a monomer (5 , 34) . We asked, therefore, whether the cleaved forms of DG arose by modification of an existing SEA domain in the DG sequence or whether it arose by insertion of an SEA module into the DG gene prior to the emergence of vertebrates. To address this question, we tested for the presence of an SEA module in the DGs of invertebrate species. Figure 5 shows the alignment of DG sequences from human (Homo sapiens), frog (Xenopus laevis), sea urchin (Strongylocentrotus purpuratus), honeybee (Apis mellifera), fruit fly (D. melanogaster), mosquito (Anopheles gambiae), and nematode (C. elegans). Above these sequences is the predicted secondary structure of the human DG SEA domain, based on our modeling in Fig. 3 . Below these sequences are examples of secondary structure predictions for the fruit fly and nematode DG sequences. The alignment shows some sequence conservation between all of these homologs in the regions corresponding to the SEA module of the human DG. More importantly, the secondary structural analysis of the DG homologs, including that of the Drosophila and C. elegans proteins, strongly predicts the structural elements of an SEA module (Fig. 5A , bottom rows). Interestingly, the two cysteines (C669 and C713) that we modeled in Fig. 3D as forming a disulfide bond are conserved in all DG homologs.


Figure 5
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  		Figure 5. Sequence alignment and structural predictions of DG homologs. A) Sequences for DG from human (H. sapiens), frog (X. laevis), sea urchin (S. purpuratus), honeybee (A. mellifera), mosquito (A. gambiae), fruit fly (D. melanogaster), and worm (C. elegans) were aligned using ClustalW. Residues are color coded according to conservation (red, completely conserved residues; cyan, identical residues; yellow, similar residues). Structures above the alignment are the secondary structure elements of DG as modeled in Fig. 3 (Blue arrows, β-sheets; red cylinders, {alpha}-helices). The two conserved cysteines are numbered. Below this alignment are the secondary structure predictions for the DG of fruit flies and nematodes, using the SAM-T06 algorithm, showing predicted {alpha}-helical (H, red), β-sheet (E, blue), and random coil (C) structures. B) Protein extracts from multiple species were probed with a monoclonal antibody (MANDAG2) against a broadly conserved sequence within the β-DG cytoplasmic domain. DG was detected at ~43 kDa in extracts from humans and sea urchin, indicating constitutive DG cleavage. Bands of ~46 and 100 kDa were observed in fruit flies, indicating partial cleavage of the DG precursor. No bands were observed in the honeybee (A. mellifera) extract, predictably, because the MANDAG2 antibody epitope is not conserved in the honeybee gene sequence.

The sequence comparison in Fig. 5A also shows the presence of the consensus cleavage site in vertebrates, and potential cleavage sites in sea urchins and honeybees, based on the presence of a glycine-serine bond between the β2 and β3 sheets. Fruit flies, mosquitoes, and nematodes appear to lack this consensus cleavage site, although a glycine-threonine bond exists in fruit flies at the beginning of the β3 sheet, which might allow some cleavage. To directly test for cleavage of the DG precursor in these organisms, a broad specificity monoclonal antibody for the β-DG subunit, MANDAG2 (35) was applied in immunoblots of protein extracts from a variety of organisms. This antibody was found to detect DG in vertebrates, sea urchins, and fruit flies, but not honeybees or mosquitoes (Fig. 5B and data not shown). This blot shows the existence of a unique ~43-kDa band in the extracts of human and sea urchin DG, reflecting complete cleavage of the DG precursor in these species. Interestingly, two bands were observed in the fruit fly extract, at ~46 and 100 kDa, indicating the partial cleavage of the DG precursor. From these analyses, we conclude that the SEA domain exists in all identified homologs of the DG gene, and that this domain likely exhibits SEA domain proteolysis in insect species such as honeybees and fruit flies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DG as an SEA protein
SEA domains were first recognized as protein modules in 1995, based on structural predictions (29) and have now been identified in many proteins with diverse functions. Although the ancestry of SEA proteins has been investigated, DG has not been assigned to this class of proteins (e.g., see ref. 32 ). Here, we provide multiple lines of evidence that place DG among the family of SEA proteins. Structural analyses show that DG possesses sequence homology with known SEA domain proteins, and both secondary and tertiary structural predictions correspond well with known SEA domain structures. Most convincingly, biochemical evidence presented here reveals a conserved mechanism of SEA domain proteolysis shared by DG and the known SEA domain protein, Muc1 (30) . In addition, the SEA domain structure of DG modeled here provides a clear explanation for the stability of the {alpha}/β-DG dimer at the cell surface. The interlacing of the 4 β sheets, two from each subunit, creates a large surface area to support strong interactions between the two subunits.

The SEA domain of DG determined here corresponds precisely to the previously undefined region of DG shown in Fig. 1B ; it starts immediately after the sequences designated as a cadherin-like domain (36) and proceeds to within 40 amino acids of transmembrane domain. Our combined analyses have permitted the construction of a detailed three-dimensional structural model for this last undefined region of the extracellular DG molecule. The positions of the {alpha}-helices, particularly {alpha}3 and {alpha}4, are the least certain portion of this model. Several of the template-free models predict a parallel alignment of the {alpha}2 and {alpha}3 helices, and this configuration remains possible (Supplemental Fig. S1). Interestingly, our model predicts a disulfide bond between cysteines 669 and 713 that forms a loop comprising the {alpha}3 and {alpha}4 helices and the β4 sheet. These two cysteines have recently been identified as structural necessities for DG cleavage, presumably through disulfide bond formation (13) . These cysteines are conserved in all DG homologs, indicating an important role in stabilizing the C-terminal half of the SEA domain, whether or not this domain is cleaved. Sequence conservation among DG homologs falls off immediately after cysteine 713, suggesting that the disulfide bond marks the end of this module.

Evidence for DG as the ancient precursor of SEA mucins
DG has a molecular composition that is evident in many transmembrane molecules classified as "mucins," which typically bear a high density of O-linked sugars but not necessarily a common molecular ancestry (32) . We now show here that DG, like many mucins, also possesses a SEA domain between its mucin and transmembrane domains and that DG shares with many mucins the constitutive SEA domain cleavage that leaves the mucin domain tethered to the cell surface through noncovalent interactions with a transmembrane subunit.

The SEA domain that we identify within the human DG molecule is evident in all DG homologs; sequence comparison shows some conservation within this region, and structural analysis shows the strong conservation of secondary structure features. We observe, by β-DG detection, constitutive cleavage of the DG in vertebrates and sea urchins, and partial cleavage in fruit flies. These cleavage events could be predicted by sequence analysis at the cleavage site. By this same analysis, the DG of honeybee is also predicted to be cleaved, because it possesses the required glycine-serine peptide bond and the constrained sequences at the junction of the β2 and β3 strands. From these observations, we conclude that the SEA domain and SEA domain proteolysis existed in the DG molecules before the divergence of vertebrates and insects. Interestingly, partial DG cleavage is observed in fruit flies, which can possibly be attributed to the serine-threonine bond present at the β2-β3 junction in the sequence of the fruit fly DG (Fig. 5) , becoming a naturally occurring example of the partial cleavage observed with the S654T mutant of the human DG (Fig. 4) .

The phylogeny of mucins has been recently investigated, and it was reported that most known homologs of SEA mucins are confined to mammals, with only Muc16 having homologs in nonmammalian vertebrates, and their ancestors of origin are uncertain (32) . Prior to our observations for DG, all of the known SEA proteins that exhibit cleavage at the sequence GSVVV or GSIVV were evident only in mammals (32) . Therefore, DG now appears as the most ancient of all known SEA proteins that exhibit this distinctive cleavage, suggesting that this cleavage mechanism could have evolved first within DG. The identification of a SEA domain in the DG of fruit flies and nematodes also places DG among the most ancient representatives of SEA mucin-like proteins. Thus, DG could be the ancestral precursor of all SEA mucins, explaining the many similarities of composition shared by these diverse molecules.

Mechanism of cleavage
Our data point to autoproteolysis as the mechanism of DG cleavage, based on concurrence with the detailed mechanism recently described for Muc1 SEA domain cleavage (30 , 33) . Sequence identity is found at the cleavage sites of Muc1 and DG, and structural similarities are maintained in the Muc1 and DG SEA domains. Most important, mutation analysis of DG shows that the basic structural determinants of cleavage are identical to those of Muc1, including the requirement for a serine, threonine or cysteine at the cleavage site. Cleavage is altered by widespread mutations within the DG SEA domain, but it is not altered by mutations of DG outside this domain, including deletion of the entire cytoplasmic and transmembrane domains and deletion of the mucin domain (2) , and broad-spectrum protease inhibitors fail to block this cleavage (11) .

Cell-free translations of DG, showing cleavage early during synthesis, mimic results for Muc1 (37) and support an autoproteolytic mechanism. However, an apparent divergence is observed as the cleavage of DG in cell-free translations is enhanced in the presence of microsomal membranes, whereas cleavage of Muc1 takes place independent of microsomes (37) . These results indicate that translocation through the ER membrane is important for DG cleavage. Translocation through the membrane is likely required for correct folding of the DG SEA domain, but it may also permit additional modifications of the protein required for cleavage. N-linked glycosylation has been proposed to facilitate DG precursor cleavage through effects on protein folding (15) , although this result has been contradicted (38) . Our data in cell-free assays show that a tripeptide inhibitor of N-linked glycosylation blocks the cleavage of the DG precursor (Fig. 2A , lane 3), indicating that N-linked glycosylation may have an effect on folding and cleavage. In addition, the disulfide bond predicted in our model appears to be required for cleavage of the DG SEA domain (13) , and it is established that disulfide bond formation takes place in the oxidizing lumen of the ER (39) . A corresponding disulfide bond is not evident in other cleaved SEA mucins, including that of Muc1, making this bond a distinct attribute of the DG SEA module and its cleavage. Exactly how this disulfide bond facilitates DG cleavage is uncertain.

Significance of SEA domain cleavage to DG function and disease
The functional significance of DG precursor cleavage has been debated. One likely role of cleavage is to permit shedding of the {alpha}-DG subunit. Shedding has been attributed to the secondary cleavage of β-DG by metalloproteinase action; however, shedding also occurs in the absence of direct cleavage of either the {alpha}- or β-subunit (11) . In mucins, shedding has been postulated as a mechanism for creating a ligand-receptor relationship between the subunits (40) or for playing more specialized roles in signaling at mucosal surfaces (30) . For DG, it has been postulated that cleavage permits independent functions of the {alpha}- and β-subunits (41) . We show here that DG’s laminin-binding capacity is enhanced by mutations that block cleavage. This result indicates that cleavage of DG may have evolved as a mechanism to modulate DG’s ligand-binding properties. Precisely how this cleavage modulates glycosylation is uncertain but likely alters the recognition of the {alpha}-DG portion by glycosyltransferases, either through effects on conformation or by allowing dissociation of the two DG subunits.

The functional significance of DG cleavage into the {alpha}/β heterodimer has been previously addressed experimentally and resulted in diverging conclusions (14 , 15 , 41) . Consistent with our observations, a DG monomer, created by deletions within the extracellular domain of β-DG, was found to function like the wild-type DG in assays of laminin assembly at neuromuscular junctions (41) . However, in another study, expression of a cleavage-defective mutant in transgenic mice resulted in a form of muscular dystrophy, hypothetically caused by reduced glycosylation of the DG monomer (14) . The apparent discrepancy between these works can possibly be explained by the fact that the S654A mutant DG expressed in transgenic mice also included a FLAG epitope tag at the N-terminal globular domain (14) . This domain has more recently been identified as the recognition site for the glycosyltransferase LARGE, and mutations within this domain can block the binding of LARGE and obstruct the functional glycosylation of {alpha}-DG (9) . In light of results presented here, we propose that the functional state of a monomeric DG in vivo remains an open question. Autoproteolysis of DG is not likely to be prevented by chemical inhibitors, but cleavage-defective mutants may have potential as gene therapy agents, augmenting DG function through stabilization of the DG molecule at the cell surface and enhancing its laminin-binding capacity.


   ACKNOWLEDGMENTS
 
This work was supported by National Institutes of Health grant R01 CA109579 (to J.L.M.) and a fellowship from the Canadian Heart and Stroke Foundation (to A.A.). The authors thank Patrick Hardy, Lynn Weir, Jarnail Singh, Yoko Itahana, and Jay Seo for technical assistance. We thank Maria Luisa Oppizzi, Jimmie Fata, and Heidi Feiler for helpful comments. We thank Kevin Campbell for the gift of antibodies, and Fred Wilts, Jessica Candy, Yury Goltsev, and Thomas Vaccari for gifts of invertebrate animals or protein samples. We also thank Drs. Jinbo Xu and Yang Zhang for creating two of the models shown in Supplemental Fig. S1, using the RAPTOR and I-TASSER methods, respectively, and Dr. Jinhui Ding for her assistance creating the Rosetta model.

Received for publication March 25, 2007. Accepted for publication August 30, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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