©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of Cranin, a Laminin Binding Membrane Protein
IDENTITY WITH DYSTROGLYCAN AND REASSESSMENT OF ITS CARBOHYDRATE MOIETIES (*)

Neil R. Smalheiser (§) , Edward Kim

From the (1)Department of Pediatrics, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cranin was described in 1987 as a membrane glycoprotein expressed in brain and many other tissues, which binds laminin with high affinity in a calcium-dependent manner. Dystrophin-associated glycoprotein (``dystroglycan'') is a laminin-binding protein cloned in 1992 whose relation to cranin has remained uncertain. Here we describe the purification of cranin to homogeneity from sheep brain, show cranin to be a form of dystroglycan, and localize the N terminus of -dystroglycan to amino acid residue 654. We find that brain -dystroglycan is tightly associated with membranes, and localizes to regions of synaptic contact as assessed by immunocytochemistry of rat cerebellum. Brain -dystroglycan expresses high mannose/hybrid N-linked saccharides, terminal GalNAc residues, and the HNK-1 epitope. Although dystroglycan has previously been presumed to be a proteoglycan, the amino acid sequence, pI, O-sialoglycoprotease susceptibility, lectin-binding profile, and laminin-binding properties of brain dystroglycan are more typical of mucin-like proteins. Furthermore, using CHO mutant cell lines deficient in xylosyltransferase and galactosyltransferase I, which are required for glycosaminoglycan biosynthesis, it is shown that chondroitin sulfate and heparan sulfate are not critical for laminin binding, and indeed are apparently not expressed at all in dystroglycan from CHO cells.


INTRODUCTION

Dystrophin-associated glycoprotein, or dystroglycan, is synthesized as a large precursor protein which is cleaved into two distinct proteins, -dystroglycan and -dystroglycan, expressed widely in many tissues(1, 2) . -Dystroglycan resides on the extracellular face of the cell surface associated with -dystroglycan (3), which in turn spans the membrane and binds directly to dystrophin (4) or utrophin (5) intracellularly. Dystroglycan appears to be important for maintaining normal muscle integrity, and it has been proposed that loss of dystroglycan from the muscle surface in Duchenne/Becker and other congenital muscular dystrophies is one of the primary events leading to muscle injury in these diseases(6) . Recently it has been proposed that dystroglycan mediates agrin-induced clustering of ACh receptors at the neuromuscular junction(7, 8, 9, 10, 11) . Dystroglycan is also expressed in brain(12) , where its mRNA largely co-localizes with dystrophin and utrophin(13) . Interest in brain dystroglycan has been greatly stimulated by the observations that cognitive deficits and mental retardation occur in many patients with Duchenne/Becker and other dystrophies(14) , that matrix proteins play important roles in neuronal migration and axonal outgrowth(15) , that dystrophin is enriched at postsynaptic densities in the central nervous system(16, 17) , and that the gene for dystroglycan in mouse maps closely to the loci of several genetic neurological diseases(2, 13) .

Cranin was described in 1987 as a heterogeneous membrane glycoprotein expressed at low abundance in brain and many other tissues, which binds laminin with high affinity in a calcium-dependent manner (18). When Ibraghimov-Beskrovnaya et al.(1) cloned the cDNA of dystroglycan, they noted its similarities to cranin, and suggested that the two proteins might be related or identical. However, the relation between these proteins has remained uncertain. For example, while cranin was characterized as primarily an integral membrane protein(18, 19) , -dystroglycan has been reported to be variously an integral membrane component(20) , a peripheral membrane component(21) , or even freely soluble in brain(12) . O-Linked saccharides on both proteins have been proposed to be critical for binding to laminin(4, 19) . However, cranin was described as a mucin-like protein which binds in a sulfatide-like manner to the E3 domain of laminin (19); in contrast, dystroglycan has generally been presumed to be a proteoglycan which binds in a heparin-like manner to the same domain (4, 12). In the present paper, we describe the purification of cranin to homogeneity from sheep brain, show that cranin is a form of dystroglycan, and provide new biochemical analyses that address some of these questions. Some of these results have been presented in preliminary form(22) .


EXPERIMENTAL PROCEDURES

Purification of Cranin

The procedure previously described for embryonic chick brain (19) was scaled-up for batches of 36 adult sheep brains. Brains were obtained at a local slaughterhouse and kept on ice for 2 h before homogenization of whole brains or gray matter in a Waring blender (low setting 30 s, high setting 1 min) in cold 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 10 mMN-ethylmaleimide, pH 7.6, adjusted with 6 N HCl, a ratio of 1.6 liters of buffer for each 12 brains. Crude membranes were pelleted (GSA rotor, 20,000 g, 1 h, 4 °C), rinsed with an equal volume of fresh 10 mM Hepes, 0.5 M NaCl, 6 M urea, pH 7.4, adjusted with 5 N NaOH, then diluted 4-fold with 10 mM Hepes, pH 7.8, and spun down again. Membranes were solubilized in 4 volumes of 2% Triton X-100 in TEA()buffer (0.01 M triethanolamine, 137 mM NaCl, 1 mM CaCl, 1 mM MgCl, pH 7.4, adjusted with 5 N NaOH) containing 5 mg/liter aprotinin, on ice for 6-8 h. After pelleting again, the supernatant was incubated batch-wise with DEAE-Sepharose CL-6B beads (7.2 liters of extract with 1.5 liters of beads) at 4 °C for at least 1 day on a rocker, filtered, and rinsed on a large sintered glass funnel, and eluted with 4.5 liters of TEA buffer containing 0.5 M NaCl, 0.1% Triton X-100. (Beads were regenerated with 2 M NaCl, 0.1% Triton X-100, TEA buffer, and stored in TEA buffer containing 0.05% sodium azide until reuse.)

The DEAE eluate was adjusted to 1 mM in MnCl, and passed in a continuous loop over a ConA-lectin affinity column (100 ml, Bio-Rad) for several days, rinsed until a plateau was reached by absorbance of the pass-through at 280 nm, and eluted slowly overnight with 300 ml of 0.4 M -methylmannoside, 0.1% Triton X-100, TEA buffer. The ConA eluate was concentrated 10-fold using a Spectrum stirred cell and Type C membrane (cutoff = 50 kDa), then diluted 10-fold in 175 mM Tris, pH 7.5, and passed in a continuous loop over a Jacalin lectin affinity column (10 ml, Vector Labs) for 2 days. After rinsing to a plateau, sample was eluted overnight in 30 ml of 0.8 M melibiose, 0.1% Triton X-100, TEA buffer. The high concentrations of sugars and slow elution times were essential to obtain an optimal yield, because of the very tight binding of cranin to the lectin columns.

The Jacalin eluate was concentrated to 2 ml on Centriprep 30 filters (Amicon), diluted to 30 ml in TEA buffer, and concentrated again, to reduce the concentration of free sugar, then treated three times with 0.5 ml of Bio-Beads SM2 (Bio-Rad) to reduce the detergent concentration. Eluate was mixed batch-wise with laminin affinity beads (3.5 ml, laminin coupled to Affi-Gel beads with a commercially available hydrazide kit (Bio-Rad)) overnight at 4 °C. Beads were rinsed and eluted with 12 ml of 1 M NaCl, 10 mM EGTA, 10 mM EDTA, 0.1% Triton X-100, TEA buffer. This first ``high salt/chelator'' eluate was concentrated to 100 µl, diluted back to 10 ml in TEA buffer, and placed again over laminin beads, rinsed, and the ``calcium chelator'' eluate was obtained in 12 ml of 10 mM EGTA, 10 mM EDTA, 0.1% Triton X-100, TEA buffer. The final eluate was concentrated to 100 µl and stored at -70 °C in 10-µl aliquots. Often, the first laminin bead pass-through was put over laminin beads and eluted twice again, to obtain additional cranin. (Laminin beads were regenerated in buffer containing 1 M NaCl, 0.1% Triton X-100, TEA buffer, and stored in TEA buffer with 0.05% azide.)

Electrophoretic Methods, Ligand-blotting Assay, and Immunoblotting Assays

In general, these methods were performed as described previously(18, 19) . Proteins were separated on 7.5% SDS-PAGE gels under reducing conditions, transferred to Immobilon-P membranes, and blocked overnight in 2% BSA, 0.1% Nonidet P-40, TEA buffer at 4 °C. In ligand-blotting assays, blots were incubated with laminin (from murine EHS sarcoma, Life Technologies, Inc.; 0.2 µg/ml in 1% BSA, TEA buffer), rinsed, incubated with an affinity-purified polyclonal anti-laminin antiserum (1:5,000), rinsed, incubated with peroxidase-conjugated anti-rabbit IgG (1:5,000), rinsed, and visualized with chemiluminescence (ECL, Amersham) usually with exposures of 1 min or less. As negative controls, laminin was omitted, or blots were incubated with laminin in the presence of 5-10 mM EGTA. In immunoblot assays, blots were blocked overnight, incubated in the presence of primary antibodies diluted in 1% BSA, TEA buffer for 1-2 h at room temperature, rinsed, incubated in the presence of secondary antibodies (usually diluted 1:5,000 in 1% BSA when the primary antibody was polyclonal, and 1:1,000 when the primary antibody was monoclonal), rinsed, incubated in avidin-biotin conjugate (Vectastain Elite ABC kit, Vector Labs, 1:4 to 1:10), rinsed, and visualized either with chemiluminescence or with 3,3`-diaminobenzidine (0.3 mg/ml) and HO (30%, 8 µl/100 ml).

Isoelectric Focusing and Two-dimensional Electrophoresis

These were carried out using a Protean II xi cell (Bio-Rad), following the manufacturer's recommended protocols and solutions exactly, with the exception that isoelectric focusing tube gels were cross-linked with 1.6% bis-acrylamide instead of 0.8%. Also, ampholytes used were Pharmalyte brand (Sigma, pH 4-6.5 and 3-10) in place of Bio-Lyte (Bio-Rad). Purified native cranin (5 µl) was mixed with isourea solution (25 µl), with or without two-dimensional SDS-PAGE standards (Bio-Rad) added to provide an internal pI calibration. Alternatively, cranin (4 µl) was denatured with 4 SDS-PAGE buffer (1 µl, final concentration 1.6% SDS), placed on a boiling water bath for 3 min, and cooled before adding isourea solution. To resolve embryonic day 14 chick brain ConA-enriched membrane extracts(19) , solid urea (24 mg) and dithiothreitol (5 µl, final concentration 1%) were added to the extract (50 µl). To resolve soluble (not membrane-bound) chick brain proteins, supernatant from brain homogenates was partially enriched in cranin by passage over DEAE and ConA columns(19) , then eluate (10 µl) was mixed with urea (5 mg), 3-10 ampholytes (3 µl), and two-dimensional PAGE standards (Bio-Rad, 25 µl in isourea solution).

Lectin-binding Assays

For routine lectin-blotting assays, blots were blocked overnight as above, incubated in biotinylated lectin (from Vector or Sigma; 0.5 µg/ml) in TEA buffer, rinsed, incubated in avidin-biotin conjugate, and visualized with 3,3`-diaminobenzidine and HO. However, this method gave high backgrounds for certain lectins because of direct binding to BSA present in the blocking and rinse solutions. In order to compare the relative binding of a large number of different lectins, the more sensitive and generally applicable method of Gravel et al. (24) was adopted, in which blots were blocked in 0.5% Tween 20, TEA buffer for 30 min with rocking at room temperature; lectin-containing and rinse solutions all contained 0.5% Tween 20, TEA buffer; and bands were visualized with chemiluminescence, dipping all treatment groups together and holding exposure times constant at 30 s.

In one series of experiments, lectin-binding intensities of untreated cranin were compared to that of neuraminidase-digested cranin: cranin (9 µl) was added to 135 µl of TEA buffer, heated to 85 °C for 5 min, adjusted to 10 mM in CaCl, and digested with neuraminidase (from Arthrobacter ureafaciens, Sigma, 1 unit/ml final concentration) for 2 h at 37 °C, stopped by adding an equal volume of 4 SDS-PAGE buffer, plus 45 µl of 11% dithiothreitol, boiled, and loaded onto 9 lanes of a SDS-PAGE gel. These incubation conditions removed 80% of sialic acid residues as assessed by the induced decrease in Mackia amurensis II lectin binding. In a second series of experiments, lectin-binding intensities were compared between samples of cranin that had been incubated with N-glycanase, and control samples incubated without enzyme. Samples of cranin were reduced and denatured by adding dithiothreitol and sodium dodecyl sulfate (1% final concentration of each), heated to 85 °C for 5 min, diluted 10-fold in 20 mM EDTA, 1% Nonidet P-40, TEA buffer, and incubated for 16 h at 37 °C, in the presence versus absence of recombinant N-glycanase (Genzyme, 6 units/ml final concentration). Reactions were stopped by adding an equal volume of 4 SDS-PAGE buffer plus dithiothreitol. These incubation conditions removed 80% of N-linked saccharides as assessed by the induced decrease in ConA-lectin binding.

Generation of Monoclonal Anti-dystroglycan Antibodies

Peptides were synthesized as the free acid corresponding to human dystroglycan amino acid residues 572-604 on an ABI model 430A peptide synthesizer using Fmoc/NMP/HObt chemistry. Peptides were cleaved from resin with trifluoroacetic acid and subsequently lyophilized, dissolved in water at 10 mg/ml, emulsified with a 4-fold excess of Freund's complete adjuvant, and injected intraperitoneal or subcutaneously into a mouse; subsequent injections were made every three weeks in Freund's incomplete adjuvant. The test bleed antiserum recognized the peptide by ELISA assay and purified cranin by immunoblotting. Spleen cells were fused with mouse SP 2/0 myeloma cells(25) ; hybridomas were screened for their ability to recognize the peptide in the ELISA assay, to stain purified cranin on immunoblots, and to recognize dystroglycan within fixed frozen sections of adult rat cerebellum. Monoclonal antibody 6C1, of the IgM class, was positive in all three assays.

Immunocytochemistry

CHO wild-type or mutant cells were plated on glass coverslips (Lab-Tek 4 well chambers, Nunc) in 10% fetal calf serum, F-12 medium, plus gentamicin, and incubated overnight at 5% CO, 37 °C. Cells were briefly rinsed in TEA buffer, then fixed in 3.7% formaldehyde in TEA buffer for 20 min, room temperature; rinsed again, and permeabilized and bleached in 0.3% HO in MeOH for 20 min at room temperature. Cells were rinsed again, blocked for several hours in 10% horse serum, incubated in 6C1 (full-strength hybridoma conditioned medium) for at least 1 h, rinsed twice in 1% serum or 3% BSA, incubated in secondary antibody (biotinylated anti-mouse Ig, Vector, 1:1,000) for 1 h, rinsed, incubated in avidin-biotin conjugate (1:3) for 1 h, rinsed, and visualized with 3,3`-diaminobenzidine and HO. As negative controls, an irrelevant monoclonal antibody (``1A5'') was substituted which did not recognize dystroglycan peptide 572-604 by ELISA assay, or primary or primary/secondary antibodies were omitted.

Perfusion and post-fixation of rats with 2% paraformaldehyde/periodate/lysine fixative, infiltration with sucrose, and sectioning of adult rat brain was performed essentially as described(26) . Sections were cut at 45 µm and reacted as floated sections. After bleaching in 0.3% HO, 30 min, sections were blocked in 5% horse serum, 0.25% Triton X-100, phosphate-buffered saline, for several hours. Sections were incubated with 6C1 (full-strength conditioned medium or diluted up to 1:50) overnight at 4 °C with gentle rocking, rinsed, incubated with biotinylated anti-mouse Ig (rat adsorbed, Vector, 1:200), 1.5 h, rinsed, incubated with avidin-biotin conjugate (1:2) for 1 h, rinsed, and visualized with Very Intense Purple (Vector Labs). As negative controls, a variety of other IgG and IgM antibodies were used in place of 6C1, or hybridoma feeding medium was used.


RESULTS

Purification of Cranin

Beginning with 36 sheep brains per preparation, 100 µg of purified cranin was obtained (). Silver staining revealed a broad 115-120 kDa band plus a doublet at 43 kDa (Fig. 1). The 115-120 kDa band corresponded to cranin as detected by ligand-blotting assay of this sample, while the 43-kDa doublet did not bind laminin in this assay and appears to have co-purified with the larger band. Ligand blotting was the most sensitive method of detecting cranin, since a strong band was routinely observed using 0.5-1% of a preparation in this assay, whereas silver staining required loading 5% of a preparation on a lane to see bands reliably (Fig. 1). The 115-120-kDa band stained heterochromatically (blue) with Stains-all, but failed to stain visibly with Coomassie Blue, even when 20% of a preparation was loaded on a single lane, which showed good staining of the 43-kDa doublet.


Figure 1: Purified cranin. A sample of purified cranin (5% of one 24-brain preparation) was separated on a 7.5% SDS-PAGE gel under reducing conditions and silver stained, revealing a prominent band at 115-120 kDa and a doublet at 43 kDa. Molecular mass markers (Sigma) are, from top, 205, 116, 97, 66, and 45 kDa.



Purified cranin was transferred to Immobilon-P membranes; the 110-kDa band was digested with endo-LysC peptide mapped by reverse phase high performance liquid chromatography, and the three most hydophobic peaks subjected to amino acid sequencing (). Two nonoverlapping peptides each gave sequences identical (except for uncertain residues) to the published sequences of rabbit, human, and mouse muscle dystroglycan(1, 2, 13) . As well, the two bands at 43 kDa were subjected to N-terminal sequencing (), and were again identical (except for uncertain residues) to the published sequences of rabbit, human, and mouse dystroglycan. Furthermore, the 110-kDa band was specifically recognized by anti-dystroglycan peptide antibodies (see below). Purity of the cranin band was confirmed by two-dimensional SDS-PAGE, revealing a single band co-migrating with cranin as assessed by silver staining (Fig. 2), ligand-blotting assay, lectin blotting, and immunoblotting (see Fig. 5, below). These findings demonstrate that cranin is a form of dystroglycan. The data also identify the N terminus of -dystroglycan as amino acid residue 654.


Figure 2: Profile of cranin on two-dimensional PAGE gels. An aliquot of cranin (eluted from laminin affinity beads in 1 M NaCl, 10 mM EGTA, 10 mM EDTA, TEA buffer) was mixed with two-dimensional SDS-PAGE standards (Bio-Rad) to provide an internal pI calibration, subjected to isoelectric focusing in 8 M urea containing CHAPS and Nonidet P-40, followed by electrophoresis by SDS-PAGE on a 7.5% gel and silver staining. The position of cranin (arrow) relative to conalbumin (A, pI = 6.0, 6.3, 6.6), bovine serum albumin (B, pI = 5.4-5.6), and bovine muscle actin (C, pI = 5.0, 5.1) indicates a heterogeneous pI for cranin, ranging from 5.3 to 6.0, and centered at 5.7. Molecular weight markers are at right.




Figure 5: HNK-1 immunoreactivity. Aliquots of cranin were subjected to two-dimensional PAGE exactly as described in the legend to Fig. 2, followed by transfer to Immobilon membranes and immunoblotting using HNK-1 antibody as described under ``Experimental Procedures.'' Blots were incubated in HNK-1 hybridoma conditioned medium (diluted 1:30), biotinylated anti-mouse IgM (1:5,000), avidin-biotin conjugate (1:5), and visualized with diaminobenzidine and HO. Cranin is strongly positive, whereas the Bio-Rad standards and molecular weight markers are not visualized (cf. Fig. 2). No bands were observed when primary antibody was omitted, or when a different IgM antibody (CD15 or Le) was employed.



The pI of purified cranin was ascertained by two-dimensional SDS-PAGE. Cranin exhibited a broad, heterogeneous pI profile from 5.3 to 6.0, centered at 5.7 (Fig. 2). The pI values were similar whether cranin was detected by silver staining, lectin-blotting, ligand-blotting, or immunoblotting assays (e.g.Fig. 5below); whether isoelectric focusing was carried out in the presence or absence of urea; and whether pH gradients were assessed by using pI protein standards mixed with cranin (Fig. 2) or by slicing isoelectric focusing gels and measuring the pH values directly. Even when cranin was fully dissociated from the 43-kDa proteins and denatured with boiling in SDS-PAGE buffer under reducing conditions prior to carrying out isoelectric focusing in the presence of 8 M urea, the pI was still centered on 5.7. Furthermore, a similar pI value was obtained when ConA-enriched membrane extracts (19) were employed instead of purified cranin, and when embryonic day 14 chick crude brain extracts were tested (both proteins enriched from the supernatant and from the pellet of the crude homogenate were tested). Finally, to verify that a more highly acidic glycoprotein of similar size could be resolved in this system, partially purified membrane extracts were separated and immunoblotted to detect the MAb 4/199 antigen(25) ; the pI of this antigen was 4.9-5.6, centered on 5.2, in agreement with our previous findings using a different isoelectric focusing apparatus(25) .

Thus, cranin/-dystroglycan appears to be only mildly acidic, which is in keeping with its elution from DEAE columns at 0.5 M NaCl(18) . The pI value of 5.7 is much less acidic than the value of 3.7 previously reported not only for 156-kDa muscle dystroglycan (27) but for 120-kDa soluble chick and bovine brain dystroglycan as well(12) . We do not currently have an explanation for this discrepancy, although it should be noted that chick and bovine brain dystroglycan were also reported to elute from DEAE columns at 0.5 M NaCl(12) , which might not be expected if it were extremely acidic.

Saccharide Modifications of Cranin

N-Linked Saccharides

The amino acid sequence of rabbit and human dystroglycan predicted by cDNA cloning exhibits 3 potential consensus sites for N-glycosylation in -dystroglycan, and a single potential site in -dystroglycan at residue 661(1, 2) . All four sites are conserved between rabbit and human species. The -dystroglycan bands bound ConA and Lens culinaris lectins by lectin blotting (Fig. 3). As well, residue 661 (asparagine) could not be reliably sequenced from either band of the 43-kDa doublet, even though the adjacent asparagine at 662 was readily detected (). Each of these findings indicate that residue 661 is glycosylated in the purified protein.


Figure 3: ConA binding sites are susceptible to endo H. Aliquots of cranin (0.5 µg) were reduced and denatured by adding dithiothreitol and sodium dodecyl sulfate (1% final concentration of each), heated to 85 °C for 5 min, diluted 10-fold in 1% Triton X-100, 0.1 M sodium acetate/acetic acid buffer, pH 6.0, with 2 mM phenylmethylsulfonyl fluoride and 50 µg of BSA added. Lane A, control, incubated for 16 h at 37 °C. B, incubated with recombinant endo H (S. plicatus, Boeringer Mannheim, 50 milliunits/ml final concentration). Samples were assayed for ConA-lectin binding by the method of Gravel et al. (24) (see ``Experimental Procedures''). Cranin/-dystroglycan (upper arrow) and the 43-kDa doublet/-dystroglycan (lower arrow) both bound ConA-lectin in lane A, whereas all staining was abolished after endo H digestion (lane B). Jacalin lectin blotting verified that cranin was still present in the endo H-digested lane, exhibiting a slightly lower apparent molecular mass by 5 kDa compared to the controls (data not shown; cf. Ref. 19). The faint band seen in both lanes represents BSA. (Another band of greater apparent mass than cranin is also seen in the lane at left, representing an abundant protein found in the Jacalin eluate; this band appears to bind laminin affinity columns weakly, and is present in the eluate when cranin is eluted from laminin beads with high salt, but usually is lost when these eluates are passed over laminin beads a second time and eluted with chelators alone.)



Lectin blotting profiles provided further information regarding N-linked saccharides on cranin/-dystroglycan. All ConA-lectin binding sites were susceptible to endo H (Fig. 3), indicating that they represent high mannose and/or hybrid structures. Several other lectins also bound cranin/-dystroglycan in a manner that was strongly decreased after N-glycanase digestion (I), indicating that they bind N-linked saccharides (without precluding that they may also bind O-linked saccharides to some extent as well). According to the manufacturer's specificity guidelines, ricin I recognizes terminal Gal or GalNAc, Dolichos biflorus recognizes terminal -linked GalNAc, Sambucus nigra recognizes Sia-2-6-Gal, Griffonia I recognizes -linked Gal or GalNAc, and wheat germ agglutinin recognizes sialic acid or GlcNAc (I). It is likely that wheat germ agglutinin recognizes sialic acid here, rather than GlcNAc, because succinylated wheat germ agglutinin bound cranin only faintly, and because neuraminidase digestion abolished binding to both of these lectins.

Taken together, these findings suggest that N-linked saccharides on cranin/-dystroglycan are primarily high mannose/hybrid structures, that at least some chains express terminal GalNAc residues (and possibly Gal as well), and that at least some of these are sialylated. In contrast, the single N-linked saccharide on the 43-kDa doublet/-dystroglycan was recognized strongly by ConA-lectin, and faintly by lens culinaris lectin and wheat germ agglutinin, but not by any of the other lectins indicated in I.

O-Linked Saccharides

The predicted amino acid sequence of -dystroglycan includes a domain extremely rich in threonine and proline, which is typical of mucin-like proteins (see below). O-Sialoglycoprotease is an enzyme from Pasteurella hemolytica that, to date, has been reported to attack only glycoproteins expressing highly clustered, sialylated O-linked saccharides(28, 29) . Cranin/-dystroglycan proved to be highly susceptible to O-sialoglycoprotease, both when the purified protein was tested and when ConA-enriched membrane extracts having cranin as a very minor component were tested (Fig. 4). Prolonged incubation with O-sialoglycoprotease had no effect upon the MAb 4/199 antigen(25) , even though it is a laminin-binding membrane glycoprotein of similar size present within the same extract (Fig. 4, C and D), and even though it expresses some O-linked saccharides(19) .


Figure 4: Susceptibility to O-sialoglycoprotease. A and B, purified cranin (1.5 µg) was incubated in TEA buffer (20 µl). Lane A, control, incubated for 1 h at 37 °C. B, incubated with O-sialoglycoprotease (Cedar Lane, Ltd., 4 µl). Samples were assayed for laminin binding via ligand blotting assay. Similar results were obtained using ConA-lectin blotting assays (not shown). C and D, embryonic day 14 chick brain membrane extracts were employed that were partially enriched in cranin through DEAE and ConA column steps (19). Extract (25 µl) was mixed with BSA (50 µg) and aprotinin (10 µg). Lane A, control, incubated for 16 h at 37 °C. B, incubated with O-sialoglycoprotease, 2 µl. In the top panel, ligand blotting assay revealed a complete loss of cranin in the treated sample, whereas, in the bottom panel, immunoblotting with monoclonal antibody 199 shows that the MAb 4/199 antigen was entirely unaffected.



Lectin-blotting profiles also provided information regarding O-linked saccharides on cranin. Six lectins bound to cranin/-dystroglycan in a manner that was not affected at all by N-glycanase digestion (I). Three of these have previously been reported to bind cranin/-dystroglycan(4, 19, 21) : Jacalin and peanut agglutinin recognize Gal1-3GalNAc disaccharides characteristic of mucins, and as expected, binding of these lectins was increased after neuraminidase treatment (I). Mackia amurensis II lectin recognizes 2,3-linked sialic acid, particularly that linked to Gal-GlcNAc. Of particular interest are three additional lectins, not previously reported: Vicia villosa lectin, which recognizes terminal GalNAc residues; Wisteria lectin, which preferentially recognizes GalNAc-Gal; and Griffonia II lectin, which specifically recognizes terminal GlcNAc residues (I).

These findings suggest that O-linked saccharides on cranin/-dystroglycan strongly express saccharide chains with terminal GalNAc residues, at least some of which are sialylated (I). It is interesting to note that the entire lectin-blotting profile described here is consistent with the presence of complex mucin-like structures having variable elongation and variable sialylation of the terminal sequence GalNAc-[Gal]-Gal-GlcNAc(30) . However, direct identification of saccharide chain lengths and sequences will have to await more detailed structural characterization (e.g. by mass spectrometry). In contrast to the prominence of O-linked saccharides on cranin/-dystroglycan, the 43-kDa doublet/-dystroglycan only exhibited faint binding of Jacalin and no binding of the other five lectins described above.

Carbohydrate Epitopes

Cranin was well stained by HNK-1 antibody (Fig. 5), which recognizes a sulfated epitope expressed on many cell adhesion molecules within the nervous system. This epitope may not be critical for laminin binding, however, since we were unable to inhibit cranin-laminin binding by preincubating blots containing purified cranin with full-strength HNK-1 conditioned medium. Although it remains uncertain whether the HNK-1 epitope in glycoproteins contains a terminal 3-sulfated glucuronic acid, it does appear to be associated with sulfated carbohydrate moieties(31) . HNK-1 antibody probably recognizes a sulfated moiety in our immunoblots as well, since HNK-1 immunoreactivity of brain membrane proteins was greatly diminished by desulfation (methanolic HCl 50-100 mM, 24 h, 25-37 °C), whereas Jacalin lectin binding was unaffected by this treatment (data not shown).

Several mucin-like recognition molecules expressed in the immune system represent ligands for the selectins(32) ; these express a set of fucose- and/or sulfate-dependent epitopes, including Le, SLe, and MECA-79(33) , which represent modifications of a lactosaminoglycan backbone in some cases(34) . However, Le, SLe, and MECA-79 antibodies did not recognize cranin on immunoblots, either before or after neuraminidase digestion. These findings are consistent with the lack of binding of Lotus and Ulex I lectins, which recognize fucose within Le structures, and with the lack of binding of a set of lectins (Lycopersicon, Datura, Solanum, and pokeweed) which typically recognize lactosaminoglycan structures (I).

Monosaccharide Composition

Neutral and amino sugars from samples of purified cranin (containing both -dystroglycan and -dystroglycan) are shown in . The composition is not remarkable, except that mannose is the most abundant sugar. This may simply reflect the presence of high mannose/hybrid structures as described above, although the presence of O-linked mannose (35) cannot be excluded at present.

Dystroglycan in CHO Wild-type versus Glycosaminoglycan-deficient Cells

We examined the expression of dystroglycan in CHO cells, since cranin has been reported to be expressed in fibroblasts(18) , and since several mutant CHO cell lines have well characterized single enzyme defects in glycosaminoglycan biosynthesis(36, 37, 38) . Ligand-blotting assays of wild-type CHO crude membrane extracts revealed a single laminin-binding band at 110-120 kDa (Fig. 6); like dystroglycan isolated from brain, this band was not removed by rinsing membranes with high salt and urea, and could be partially purified on DEAE and ConA columns in a similar fashion. No laminin-binding bands were observed if assays were carried out in the absence of calcium, or if extracts were first digested with O-sialoglycoprotease.


Figure 6: Expression of -dystroglycan in CHO cells. CHO wild-type cells (A), xylosyltransferase-deficient cells (B), and galactosyltransferase-deficient cells (C) were grown to confluency, scraped in homogenization buffer (see ``Experimental Procedures'' for sheep brain purification), and subjected to 30 strokes of a Dounce homogenizer. Crude membranes were pelleted in a Sorvall SS-34 rotor at 20,000 g, 45 min, then rinsed in high salt/urea buffer, pelleted again, and solubilized. Aliquots representing material from approximately 4 confluent 100-mm tissue culture dishes were loaded onto each lane (adjusted further so that equal amounts of protein were loaded in each lane), and analyzed by ligand blotting assay. A broad band at 110-120 kDa was observed in all three groups (large arrow). This was the only laminin-binding band detected in these extracts, since the other bands were still observed when laminin was omitted from the assay (data not shown; the highest band (small arrow) migrates at 220 kDa and presumably represents laminin B chains produced by the CHO cells).



Wild-type CHO cells (K1 strain) were grown in parallel with two mutant cell lines (a generous gift of Dr. J. Esko), one deficient in xylosyltransferase (``745'') and one deficient in galactosyltransferase I (``761'')(36, 37, 38) . Both have been characterized as failing to synthesize chondroitin sulfate and heparan sulfate. After confirming that these mutants incorporated relatively little radioactive sulfate into trichloroacetic acid-precipitable material (<15% of wild-type levels), all three lines were grown to confluency, membrane extracts were prepared, and the mobility and laminin-binding properties of these mutants were compared with wild-type cells. As shown in Fig. 6, all three cell lines expressed a single broad laminin-binding band at 110-120 kDa. The apparent size and yield of dystroglycan were similar in the wild-type and xylosyltransferase mutant, whereas the galactosyltransferase I mutant consistently expressed a higher yield than the wild-type (Fig. 6). In all three cell lines, no laminin-binding bands were observed if assays were carried out in the absence of calcium, or if extracts were first digested with O-sialoglycoprotease.

To confirm that these laminin-binding bands represented authentic dystroglycan, DEAE-enriched membrane extracts of CHO cells were immunoblotted with anti-dystroglycan antiserum (Fig. 7). Moreover, anti-dystroglycan peptide antibody 6C1 stained CHO wild-type cells (Fig. 8), both CHO mutant cell lines (data not shown) and adult rat cerebellum (Fig. 9) strongly and specifically by immunocytochemistry.


Figure 7: Immunoblotting of CHO membrane extracts with -dystroglycan antiserum. CHO cells from wild type and mutant lines were grown to confluency in parallel and homogenized. In order to obtain adequate amounts of dystroglycan for immunoblotting, crude membrane extracts from 25 100-mm dishes were rinsed with high salt and urea, solubilized, and enriched over DEAE beads with elution at 0.5 M NaCl; dystroglycan still represents only a very minor component of this eluate. Equal amounts of protein were loaded on each lane. A-C, immunoblotted with anti-dystroglycan peptide antiserum (1:200). D-F, immunoblotted with normal mouse serum as a negative control. A and D, wild-type. B and E, xylosyltransferase-deficient. C and F, galactosyltransferase I-deficient. A single broad band at 110-120 kDa is seen in each lane (arrow; cf. Fig. 6). Molecular weight markers are at left.




Figure 8: Immunostaining of CHO cells with -dystroglycan peptide antibody 6C1. Upper panel, CHO wild-type cells were immunostained as described under ``Experimental Procedures.'' Particularly strong staining was observed in a juxtanuclear region (presumably the Golgi region) within cells that were well spread. In cells that were more rounded, discrete punctate staining of the cell surface was also observed, apparently corresponding to small microvillar protrusions of membrane. Lower panel, as a negative control, no staining was observed when cells were incubated with hybridoma conditioned medium 1A5 (which did not recognize dystroglycan peptide 572-604 by ELISA assay).




Figure 9: Immunostaining of adult rat cerebellum with -dystroglycan peptide antibody 6C1. Cerebellar sections were immunostained as described under ``Experimental Procedures.'' Strong, selective staining was observed of fibers throughout the molecular layer (M). Little staining is observed within the granule cell layer (G) or white matter (W) except for some blood vessels. At higher power, in favorable sections (inset), some of the stained fibers are seen to exhibit a pattern of dots, short segments, and spines that closely follows the trajectory of climbing fibers upon Purkinje cell dendrites. This suggests that the immunoreactive product is localized to regions of synaptic contact upon the dendrites. Although one cannot resolve whether staining is presynaptic, postsynaptic or both, the findings are consistent with: (a) an in situ hybridization study (13) indicating that dystroglycan mRNA is restricted to Purkinje cells in the adult mouse cerebellum; (b) an immunocytochemical study (17) indicating that dystrophin is enriched at central nervous system postsynaptic densities in general, and upon Purkinje cell dendrites in particular; and (c) reports that dystroglycan is enriched at postsynaptic membranes in electric organ (20) and muscle (Ref. 5; see also Ref. 11). When sections were incubated with a variety of other IgG or IgM antibodies, or with hybridoma feeding medium alone, some light diffuse staining of Purkinje cell bodies was still observed, but the characteristic pattern of fiber staining within the molecular layer was absent. Also, fiber staining intensity was reduced when 6C1 (2.5 ml at 1:50 dilution) was preabsorbed with peptide 572-604 (4 mg/ml), but not when 6C1 was preabsorbed with a different peptide (data not shown).




DISCUSSION

Dystroglycan is a cell surface glycoprotein which binds directly to extracellular matrix proteins and to the underlying cytoskeleton (4). Dystroglycan is expressed very widely across tissues and may be important generally in mediating or modulating many of the effects of extracellular matrix on cells. Yet dystroglycan varies widely in its association with other proteins. In different places, dystroglycan associates with laminin(39) , merosin(40) , or agrin(7, 8, 9, 11) , and may associate either with dystrophin (4) or utrophin(5) . In muscle, dystroglycan also associates with a set of so-called sarcoglycan complex proteins (3) which are either absent or are antigenically different in non-muscle tissues(4) .

Dystroglycan isolated from different tissues has different mobilities on SDS-PAGE gels ranging from 120 to 200 kDa(1, 12, 19, 20) . Since dystroglycan is encoded by a single-copy gene(2) , and since only a single mRNA and protein species has been reported to date(2) , saccharide modifications of dystroglycan are likely to underlie its tissue-specific heterogeneity. Considerable indirect evidence suggests that the O-linked saccharides on dystroglycan are critical for its high affinity binding to laminin(4, 19) . However, the functional significance of tissue-specific heterogeneity remains unclear at present, for example, brain and muscle dystroglycan both appear to bind laminin and agrin()in a similar manner(4, 7) .

Another laminin-binding membrane protein, cranin, was described in brain and other tissues several years prior to the discovery of dystroglycan(18) . In the present paper, cranin has been purified to homogeneity from sheep brain, identified as a form of dystroglycan, and characterized in several ways which extend and reassess current knowledge of dystroglycan structure and function.

Reassessing Properties of Brain Dystroglycan

In our hands, brain dystroglycan is strikingly similar to dystroglycan isolated from a modified synapse, the Torpedo electric organ(7, 20) , in that the -subunits behave as integral membrane components(18, 19) , and co-purify on ligand affinity columns with a tightly (but noncovalently) associated -dystroglycan subunit (see also Ref. 4). These findings are in marked contrast to a report by Gee et al.(1993) which described 80% of brain -dystroglycan as recoverable from ultrasupernatants of saline tissue homogenates, i.e. not membrane bound, and not associated with -dystroglycan after purification(12) . Several other discrepant findings also exist between our two laboratories (see ``Results'' and Ref. 19) and the reasons remain unclear. However, monoclonal anti-dystroglycan peptide antibody 6C1 localized dystroglycan in adult rat cerebellum along Purkinje cell dendrites (Fig. 9), in a distribution which co-localizes with dystrophin(17) . Such a pattern strongly suggests that a significant portion of dystroglycan is membrane associated, at sites of synaptic contact, in the brain in situ.

N Terminus of -Dystroglycan

Analyses of consensus sequences for proteolytic cleavage have suggested that the N terminus of -dystroglycan might lie at residues 457 (1) or 640(12) . Direct N-terminal sequencing of both 43-kDa doublet bands now reveals the site to be residue 654 (), which is somewhat surprising since it is not directly adjacent to a basic residue.

N-Linked Saccharides

The N-linked saccharides expressed on - and -dystroglycan are shown to be of the high mannose/hybrid variety (Fig. 3). These are not necessary for laminin binding(4, 19) , nor does mannan inhibit laminin binding(19) , but it is possible that they might be involved in other recognition or binding events. For example, oligomannosides have been reported to interact with cell surfaces to promote cell spreading(41) , and NCAM and L1 are known to bind to each other in cis on cell surfaces via a lectin-like domain of NCAM binding to oligomannose residues on L1(42) . In this regard, it is noteworthy that three of the four potential N-glycosylation sites in dystroglycan are located more proximally to the membrane than the mucin-like domain, and hence would be well located to interact in cis with sarcoglycan proteins (3, 6) or other proteins on the cell surface.

Other Saccharide-based Recognition Motifs

Both N-linked and O-linked saccharides on cranin/-dystroglycan are shown to bind a number of lectins strongly which recognize terminal GalNAc residues (I). It is worth noting that GalNAc-specific lectins are also known to bind selectively to the neuromuscular junction, where they identify a number of glycoproteins and glycolipids(43) . Moreover, synthetic oligosaccharides such as GalNAc-1,3-Gal-1,4-Glc are especially potent, nontoxic inhibitors of proliferation in neural cells(44) . The HNK-1 epitope, expressed on brain -dystroglycan (Fig. 5), has also been proposed to have a potential recognition/binding role(45, 46) . Finally, the presence of terminal GlcNAc residues (indicated by Griffonia II lectin binding) suggests that -dystroglycan may be an acceptor for Gal- or GalNAc-specific glycosyltransferases.

Is Dystroglycan a Proteoglycan?

Most previous discussions of dystroglycan have presumed that it is likely to be a proteoglycan. The high carbohydrate content, Alcian blue staining(4) , and low pI (3.7) of muscle dystroglycan (27) are all typical of proteoglycans. Moreover, binding of dystroglycan to matrix proteins is inhibited by heparin(4, 9, 12) . S27 muscle cells, which are deficient in synthesizing chondroitin sulfate and heparan sulfate, make a form of dystroglycan which is abnormally small and which binds agrin and laminin poorly(8, 9, 11) . Finally, the predicted amino acid sequence of dystroglycan contains two conserved Ser-Gly consensus sequences for addition of glycosaminoglycans(2) . On the other hand, at least four groups, working with both muscle and brain dystroglycan, have been unable to affect the mobility or laminin-binding properties of dystroglycan using chondroitinases, heparitinases, or keratanase(4, 7, 12, 19) , and cranin was previously reported to resist nitrous acid treatment which degrades heparin and heparan sulfate(18) . Moreover, the molecular defect in S27 cells has not been identified, and it has not been ruled out that O-linked saccharides might be generally abnormal in these cells.

An alternative view is that dystroglycan is not a proteoglycan at all, but instead resembles a typical mucin-like membrane protein(47) . Although Lasky et al.(48) have commented in passing on the mucin-like nature of sequences present within dystroglycan, it bears emphasis that dystroglycan exhibits a discrete domain extremely rich in Thr and Pro: in a stretch of 169 residues(317-485) there are 40 Thr, many of them clustered in short runs; 10 Ser; and 32 Pro, 24 of them adjacent to Thr, Pro, or Ser. Such a pattern closely resembles peptide sequences which have been shown to be good acceptors for GalNAc transferase(49) . The susceptibility of brain and CHO cell dystroglycan to O-sialoglycoprotease (Fig. 4) and its lectin-binding profile (I) are consistent with the properties of mucin-like proteins, as is the sulfatide-like manner in which cranin and certain mucin-like proteins each bind their ligands(19, 32, 50, 51) . Experiments reported here in CHO mutant cells lacking xylosyltransferase and galactosyltransferase I indicate that chondroitin sulfate and heparan sulfate are not necessary for high affinity binding of dystroglycan to laminin, and indeed these chains appear to be absent entirely from wild-type CHO cells ( Fig. 6and Fig. 7). It remains possible that dystroglycan might be a ``part-time'' proteoglycan expressing glycosaminoglycan chains in muscle. Alternatively, tissue-specific expression of mucin-like chains (52) or differing levels of sulfation of these chains (53) could provide the basis for tissue-specific differences in dystroglycan structure and function.

Future Prospects

Our data demonstrate the purification of dystroglycan from mammalian brain in amounts which should permit direct structural examination of its saccharide moieties. The high and selective susceptibility of dystroglycan to O-sialoglycoprotease, together with its lack of toxicity for living myeloid cells (29) and NG108-15 cells,()suggests exploring the use of this protease as a novel experimental tool for identifying and analyzing the functions of mucin-like proteins in neural cells. Monoclonal anti-dystroglycan peptide antibody 6C1 is, to our knowledge, the first antibody described which is suitable for carrying out immunocytochemical localization of -dystroglycan in the central nervous system. Therefore, the present studies open several new avenues of research.

  
Table: Purification of cranin from sheep brain

In each step, yields were calculated by measuring protein content of start, pass through, and eluate fractions. Recoveries were estimated by comparing the relative intensity of laminin-binding bands obtained from equal aliquots of start, pass through, and eluate fractions in the ligand-blotting assay.


  
Table: Amino acid sequencing of cranin peptides and N-terminal sequence of 43-kDa doublet

Purified cranin (100 µg) was separated on 7.5% SDS-PAGE gels under reducing conditions, transferred to Immobilon-P membranes (Millipore) as described (18), and stained with Amido Black; the 120-kDa band was cut out and digested with endo Lys-C followed by separation on reverse-phase HPLC columns as described (7), and the three most hydrophobic peaks were sequenced as described (7, 23). The upper and lower bands of the 43-kDa doublet were cut out separately and subjected to N-terminal sequencing. Initial yields of cranin peptides were 10 pmol, and for 43-kDa doublet bands were 2 pmol each. Dystroglycan residues are numbered relative to the predicted amino acid sequences of rabbit, mouse and human species (1, 2, 13). Uncertain assignments are indicated in bold face. Asterisks indicate asparagines which satisfy consensus for N-linked glycosylation.


  
Table: Lectin-blotting profile of cranin/-dystroglycan

Lectin-blotting was performed as described under ``Experimental Procedures,'' scoring band intensities on a 0-5 scale relative to ConA and Jacalin binding of untreated samples (set at 5). Binding to untreated samples was tested for all lectins on at least two occasions under these standardized comparative conditions, and most lectins which bound cranin were tested further as well. A ``strong decrease'' in lectin binding after N-glycanase treatment implies at least 50% loss of band intensity relative to incubated control sample.


  
Table: Monosaccharide composition (neutral and amino sugars)

Purified samples (50 µg in 60 µl; containing both cranin/-dystroglycan and the 43-kDa doublet/-dystroglycan) were treated with BioBeads SM (Bio-Rad) to remove excess detergent. After hydrolyzing with trifluoroacetic acid (2 M, 3 h, 100 °C), neutral and amino sugars were separated by HPLC using a CarboPac PA-1 column, quantified by peak areas, and identified relative to standards run in parallel. The GlcNAc peak was normalized to 1.00, and other peaks compared to it. Two different samples were tested; shown is the result from the sample having the smaller amount of glucose contamination.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants HD 09402 and NS 26055 and the Brain Research Foundation, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pediatrics, University of Chicago, MC 5058, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-6501; Fax: 312-702-9234.

The abbreviations used are: TEA, triethanolamine; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate; BSA, bovine serum albumin; CHO, Chinese hamster ovary.

N. Smalheiser and H. Benjamin Peng, unpublished observations.

N. Smalheiser, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Jeffrey Esko (University of Alabama at Birmingham) for gifts of CHO wild-type and mutant cell lines; Dr. Eugene Butcher (Stanford University, Stanford, CA) for a gift of MECA-79 antibody; and Dr. Barbara Collins (Michael Reese Hospital, Chicago, IL) for performing immunocytochemistry on rat cerebellum. Thanks also to Drs. Steven Rosen, Nancy Schwartz, and Justin Fallon for helpful advice. Monosaccharide analyses were performed at a facility supervised by Dr. Adriana Manzi (University of California at San Diego), and amino acid sequencing at a facility supervised by Dr. John Leszyk (Worcester Foundation, Shrewsbury, MA).


REFERENCES
  1. Ibraghimov-Beskrovnaya, O., Ervasti, J., Leveille, C., Slaughter, C., Sernett, S., and Campbell, K.(1992) Nature355, 696-702 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ibraghimov-Beskrovnaya, O., Milatovich, A., Ozcelik, T., Yang, B., Koepnick, K., Francke, U., and Campbell, K.(1993) Hum. Mol. Genet.2, 1651-1657 [Abstract]
  3. Yoshida, M., Suzuki, A., Yamamoto, H., Noguchi, S., Mizuno, Y., and Ozawa, E.(1994) Eur. J. Biochem.222, 1055-1061 [Abstract]
  4. Ervasti, J., and Campbell, K.(1993) J. Cell Biol.122, 809-823 [Abstract]
  5. Matsumura, K., Ervasti, J., Ohlendieck, K., Kahl, S., and Campbell, K. (1992) Nature360, 588-591 [CrossRef][Medline] [Order article via Infotrieve]
  6. Matsumura, K., and Campbell, K.(1994) Muscle & Nerve17, 2-15
  7. Bowe, M., Deyst, K., Leszyk, J., and Fallon, J.(1994) Neuron12, 1-20 [Medline] [Order article via Infotrieve]
  8. Campanelli, J., Roberds, S., Campbell, K., and Scheller, R.(1994) Cell77, 663-674 [Medline] [Order article via Infotrieve]
  9. Gee, S., Montanaro, F., Lindenbaum, M., and Carbonetto, S.(1994) Cell77, 675-686 [Medline] [Order article via Infotrieve]
  10. Sealock, R., and Froehner, S.(1994) Cell77, 617-619 [Medline] [Order article via Infotrieve]
  11. Sugiyama, J., Bowen, D., and Hall, Z.(1994) Neuron13, 103-115 [Medline] [Order article via Infotrieve]
  12. Gee, S., Blacher, R., Douville, P., Provost, P., Yurchenco, P., and Carbonetto, S.(1993) J. Biol. Chem.268, 14972-14980 [Abstract/Free Full Text]
  13. Gorecki, D., Derry, J., and Barnard, E.(1994) Hum. Mol. Genet.3, 1589-1597 [Abstract]
  14. Bresolin, N., Castelli, E., Comi, G., Felisari, G., Bardoni, A., Perani, D., Grassi, F., Turconi, A., Mazzucchelli, F., Gallotti, D., Moggio, M., Prelle, A., Ausenda, C., Fazio, G., and Scarlato, G. (1994) Neuromusc. Disorders4, 359-369 [CrossRef][Medline] [Order article via Infotrieve]
  15. Letourneau, P. C., Condic, M. L., and Snow, D. M.(1994) J. Neurosci.14, 915- 928 [Medline] [Order article via Infotrieve]
  16. Kim, T-W., Wu, K., Xu, J-L., and Black, I.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 11642-11644 [Abstract]
  17. Lidov, H., Byers, T., and Kunkel, L.(1993) Neuroscience54, 167-187 [Medline] [Order article via Infotrieve]
  18. Smalheiser, N. R., and Schwartz, N. B.(1987) Proc. Natl. Acad. Sci. U. S. A.84, 6457-6461 [Abstract]
  19. Smalheiser, N. R.(1993) J. Neurosci. Res.36, 528-538 [Medline] [Order article via Infotrieve]
  20. Ma, J., Nastuk, M., McKechnie, B., and Fallon, J.(1993) J. Biol. Chem.268, 25108-25117 [Abstract/Free Full Text]
  21. Ervasti, J., and Campbell, K.(1991) Cell66, 1121-1131 [Medline] [Order article via Infotrieve]
  22. Smalheiser, N. R., and Kim, E.(1994) Soc. Neurosci. Abstr.20, 1693
  23. Fernandez, J., Andrews, L., and Mische, S.(1994) Anal. Biochem.218, 112-117 [CrossRef][Medline] [Order article via Infotrieve]
  24. Gravel, P., Golaz, O., Walzer, C., Hochstrasser, D., Turler, H., and Balant, L.(1994) Anal. Biochem.221, 66-71 [CrossRef][Medline] [Order article via Infotrieve]
  25. Smalheiser, N. R., and Collins, B. J.(1992) Dev. Brain Res.69, 215-223 [Medline] [Order article via Infotrieve]
  26. Smalheiser, N. R., and Collins, B. J.(1992) Dev. Brain Res.69, 225-231 [Medline] [Order article via Infotrieve]
  27. Yamamoto, H., Hagiwara, Y., Mizuno, Y., Yoshida, M., and Ozawa, E. (1993) J. Biochem. (Tokyo) 114, 132-139 [Abstract]
  28. Sutherland, D., Abdullah, K., Cyopick, P., and Mellors, A.(1992) J. Immunol.148, 1458-1464 [Abstract/Free Full Text]
  29. Norgard, K., Moore, K., Diaz, S., Stults, N., Ushiyama, S., McEver, R., Cummings, R., and Varki, A.(1993) J. Biol. Chem.268, 12764-12774 [Abstract/Free Full Text]
  30. Lesuffleur, T., Zweibaum, A., and Real, F.(1994) Crit. Rev. Oncol./Hematol.17, 153-180 [Medline] [Order article via Infotrieve]
  31. Field, M., Wing, D., Dwek, R., Rademacher, T., Schmitz, B., Bollensen, E., and Schachner, M.(1992) J. Neurochem.58, 993-1000 [Medline] [Order article via Infotrieve]
  32. Varki, A.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 7390-7397 [Abstract]
  33. Hemmerich, S., Butcher, E., and Rosen, S.(1994) J. Exp. Med.180, 2219-2226 [Abstract]
  34. Moore, K. L., Eaton, S. F., Lyons, D. E., Lichenstein, H. S., Cummings, R. D., and McEver, R. P.(1994) J. Biol. Chem.269, 23318-23327 [Abstract/Free Full Text]
  35. Krusius, T., Finne, J., Margolis, R. K., and Margolis, R. U.(1986) J. Biol. Chem.261, 8237-8242 [Abstract/Free Full Text]
  36. Esko, J., Stewart, T., and Taylor, W.(1985) Proc. Natl. Acad. Sci. U. S. A.82, 3197-3201 [Abstract]
  37. Esko, J., Elgavish, A., Prasthofer, T., Taylor, W., and Weinke, J. (1986) J. Biol. Chem.261, 15725-15733 [Abstract/Free Full Text]
  38. Esko, J., Weinke, J., Taylor, W., Ekborg, G., Roden, L., Anantharamaiah, G., and Gawish, A.(1987) J. Biol. Chem.262, 12189-12195 [Abstract/Free Full Text]
  39. Dickson, G., Azad, A., Morris, G., Simon, H., Noursadeghi, M., and Walsh, F.(1992) J. Cell Sci.103, 1223-1233 [Abstract/Free Full Text]
  40. Yamada, H., Shimizu, T., Tanaka, T., Campbell, K., and Matsumura, K. (1994) FEBS Lett.352, 49-53 [CrossRef][Medline] [Order article via Infotrieve]
  41. Chandrasekaran, S., Tanzer, M., and Giniger, M.(1994) J. Biol. Chem.269, 3367-3373 [Abstract/Free Full Text]
  42. Horstkorte, R., Schachner, M., Magyar, J., Vorherr, T., and Schmitz, B. (1993) J. Cell Biol.121, 1409-1421 [Abstract]
  43. Scott, L., Bacou, F., and Sanes, J.(1988) J. Neurosci.8, 932-944 [Abstract]
  44. Santos-Benito, F., Fernandez-Mayoralas, A., Martin-Lomas, M., and Nieto-Sampedro, M.(1992) J. Exp. Med.176, 915-918 [Abstract]
  45. Hall, H., Liu, L., Schachner, M., and Schmitz, B.(1993) Eur. J. Neurosci.5, 34-42 [Medline] [Order article via Infotrieve]
  46. Needham, L. K., and Schnaar, R. L.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 1359-1363 [Abstract]
  47. Shimizu, Y., and Shaw, S.(1993) Nature366, 630-631 [CrossRef][Medline] [Order article via Infotrieve]
  48. Lasky, L., Singer, M., Dowbenko, D., Imai, Y., Henzel, W., Grimley, C., Fennie, C., Gillett, N., Watson, S., and Rosen, S.(1992) Cell69, 927-938 [Medline] [Order article via Infotrieve]
  49. Elhammer, A., Poorman, R., Brown, E., Maggiora, L., Hoogerheide, J., and Kezdy, F.(1993) J. Biol. Chem.268, 10029-10038 [Abstract/Free Full Text]
  50. Imai, Y., True, D., Singer, M., and Rosen, S.(1990) J. Cell Biol.111, 1225-1232 [Abstract]
  51. Taraboletti, G., Rao, C., Krutzsch, H., Liotta, L., and Roberts, D. (1990) J. Biol. Chem.265, 12253-12258 [Abstract/Free Full Text]
  52. Baumhueter, S., Singer, M., Henzel, W., Hemmerich, S., Renz, M., Rosen, S., and Lasky, L.(1993) Science262, 436-438 [Medline] [Order article via Infotrieve]
  53. Dowbenko, D., Kikuta, A., Fennie, C., Gillett, N., and Lasky, L.(1993) J. Clin. Invest.92, 952-960 [Medline] [Order article via Infotrieve]

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