(Received for publication, August 14, 1995)
From the
Phosphacan, a soluble nervous tissue-specific chondroitin
sulfate proteoglycan, is an alternative splicing product representing
the entire extracellular domain of a transmembrane receptor-type
protein-tyrosine phosphatase (RPTP/
) that also occurs as a
chondroitin sulfate proteoglycan in brain. We have previously
demonstrated that phosphacan binds with high affinity to neural cell
adhesion molecules (Ng-CAM/L1 and N-CAM) and to the extracellular
matrix protein tenascin and that it is a potent inhibitor of cell
adhesion and neurite outgrowth. Tryptic digests of
I-labeled phosphacan contain two glycopeptides that bind
to Ng-CAM/L1, N-CAM, and tenascin. The larger of these (17 kDa) begins
at Gln-209 near the end of the carbonic anhydrase-like domain of
phosphacan/RPTP
/
, whereas a 13-kDa glycopeptide begins at
His-361 located in the middle of the fibronectin type III-like domain.
Treatment of phosphacan with peptide N-glycosidase under
nondenaturing conditions reduced its binding to the neural cell
adhesion molecules and tenascin by 65-75%, whereas
endo-
-N-acetylglucosaminidase H had no effect, and
peptide N-glycosidase treatment both decreased the molecular
sizes of the tryptic peptides to
11 kDa and abolished their
binding. Based on the amino acid sequence of phosphacan, it can be
concluded that each of the tryptic peptides contains one potential N-glycosylation site (at Asn-232 and Asn-381), and analyses of
the isolated glycopeptides demonstrated the presence of sialylated
complex-type oligosaccharides. Our results therefore indicate that the
interactions of phosphacan/RPTP
/
with neural cell adhesion
molecules and tenascin are mediated by asparagine-linked
oligosaccharides present in their carbonic anhydrase- and fibronectin
type III-like domains.
We have previously described the isolation and biochemical
properties of phosphacan, a nervous tissue-specific chondroitin sulfate
proteoglycan that is synthesized by astrocytes (Rauch et al.,
1991). Cloning of phosphacan (Maurel et al., 1994)
demonstrated that it is an alternative splicing product representing
the entire extracellular domain of a receptor-type protein-tyrosine
phosphatase (RPTP) ()named RPTP
/
(Krueger and
Saito, 1992; Levy et al., 1993; Maurel et al., 1995).
A keratan sulfate-containing glycoform (phosphacan-KS) also occurs in
postnatal brain (Rauch et al., 1991; Maurel et al.,
1994; Meyer-Puttlitz et al., 1995). Phosphacan binds
reversibly and with high affinity to the neural cell adhesion molecules
Ng-CAM/L1 and N-CAM (K
0.1
nM) and to the extracellular matrix protein tenascin (K
= 3 nM), but not to
over a dozen other cell membrane and extracellular matrix proteins
tested (Milev et al., 1994; Grumet et al., 1994).
These studies also demonstrated that phosphacan is a potent inhibitor
of neuronal and glial adhesion and of neurite outgrowth. Because of its
potential importance both as a developmentally regulated extracellular
matrix proteoglycan of nervous tissue and as the ligand-binding domain
of a transmembrane protein-tyrosine phosphatase (which also occurs in
the form of a chondroitin sulfate proteoglycan in brain; Shitara et
al., 1994), we have attempted to identify the region of
phosphacan/RPTP
/
that participates in its interactions with
nervous tissue proteins, using the glycosylated proteoglycan that is
synthesized by rat brain in vivo. Phosphacan contains an
N-terminal carbonic anhydrase-like domain followed by a single
fibronectin type III sequence and a glycosaminoglycan linkage region to
which are attached three to four chondroitin sulfate chains and (in the
case of phosphacan-KS) a similar or greater number of keratan sulfate
chains which vary in length and fine structure (Rauch et al.,
1991; Maurel et al., 1994). We have found that two tryptic
glycopeptides derived from the carbonic anhydrase- and fibronectin type
III-like domains of phosphacan/RPTP
/
bind to the neural cell
adhesion molecules and tenascin and that their interactions are
mediated by asparagine-linked oligosaccharides.
Trypsin and chondroitinase ABC digestions were
performed at 37 °C in 100 mM Tris-HCl buffer, pH 8.0,
containing 30 mM sodium acetate. A range of trypsin to
phosphacan ratios was tested, from which it was found that the optimal
conditions for generation of peptides capable of binding to Ng-CAM were
2-h digestion at an enzyme-substrate ratio of 1 to 20 (w/w) or 18 h at
a ratio of 1 to 200-400. L-1-tosylamido-2-phenylethyl
chloromethyl ketone-trypsin was obtained from Worthington, and
following digestion the enzyme was inactivated using 0.5 mMN-p-tosyl-L-lysine
chloromethyl ketone (Sigma).
For removal of asparagine-linked
oligosaccharides, phosphacan was treated for 6 h at 37 °C with
either recombinant peptide N-glycosidase (EC 3.5.1.52) or with
a mixture of peptide N-glycosidase and
endo--N-acetylglucosaminidase F
(Endo
F
) purified from Flavobacterium meningosepticum. Digestions were performed in 50 mM PBS, pH 7.4,
containing 0.5% Nonidet P-40 and 2 mg/ml heat-treated BSA, using
0.125-0.5 units of glycosidase/100 ng of proteoglycan protein (2
10
cpm). Phosphacan was also treated with
recombinant endo-
-N-acetylglucosaminidase H (Endo H)
prepared from Streptomyces lividans. Digestion with Endo H was
performed at pH 6.5 or 7.4 for 18 h in 50 mM PBS containing
0.5% Nonidet P-40 and 2 mg/ml heat-treated BSA, using 10 milliunits of
glycosidase/100 ng of proteoglycan protein. All endoglycosidases were
obtained from Boehringer Mannheim, and units are according to the
manufacturer's definitions, which differ for the three enzymes.
Tryptic (glyco)peptides prepared from I-labeled
phosphacan gave a complex pattern on an SDS-PAGE gradient gel (Fig. 1A). When this mixture was added to plastic wells
coated with chick Ng-CAM, its rat homolog L1/NILE, or rat or chick
N-CAM, two bands with apparent molecular sizes of 13 and 17 kDa were
seen on autoradiographs of SDS gels of the bound radioactivity (Fig. 1D and data not shown), and proteins of these
sizes were depleted from the unbound material (Fig. 1C). The bound 13- and 17-kDa peptides were also
seen without chondroitinase treatment and following elution and
electrophoresis in the absence of mercaptoethanol. These results are
consistent with both peptides originating from regions of phosphacan
before the first potential chondroitin sulfate attachment site at
Ser-595 (Maurel et al., 1994) and demonstrate that each
peptide binds independently, rather than as a result of disulfide
linkage to a single peptide that contains the binding site. There was
much less binding of tryptic peptides to immobilized tenascin, which
probably reflects the 30-fold lower affinity of the phosphacan-tenascin
as compared with the phosphacan-Ng-CAM interactions (Grumet et
al., 1994; Milev et al., 1994). However, the 13- and
17-kDa peptides could also be identified by SDS-PAGE and
autoradiography after elution from tenascin-coated wells.
Figure 1:
Autoradiographs
of tryptic peptides prepared from I-labeled phosphacan
and electrophoresed on a 13-cm 10-20% gradient gel (lanes A and E-H) or on a 12.5% minigel (lanes
B-D). Lanes A and B, total tryptic
peptides; lane C, peptides that did not bind to Ng-CAM; D, peptides bound to and eluted from Ng-CAM. Bound peptides
eluted from L1/NILE (the rat homolog of Ng-CAM) and from rat or chick
N-CAM and tenascin gave a pattern identical to that seen in lane D (not shown). Peptide N-glycosidase treatment of
I-labeled phosphacan isolated from 7-day postnatal (lanes E and F) or adult (lanes G and H) rat brain results in the disappearance of the 17- and
13-kDa tryptic glycopeptides and the appearance of a new band at
11 kDa (lanes F and H).
To determine the origin of these peptides within the phosphacan sequence, unlabeled proteoglycan was mixed with iodinated material, digested with trypsin, fractionated by SDS-PAGE, and transferred to a ProBlott membrane. The positions of the 13- and 17-kDa peptides were identified by autoradiography, and the excised bands were used for N-terminal microsequencing. The 13-kDa peptide gave a sequence (HEFLT DGYQDLGAILNNLIP-MSYV) beginning at His-361 of phosphacan, whereas the 17-kDa peptide sequence (QA-FILQNLLPISTDKYY) began at Gln-209 (Fig. 2A).
Figure 2:
A, amino acids 201-500 of phosphacan
(Maurel et al., 1994) showing the trypsin cleavage sites used
for generation of the 17 (filled arrowhead)- and 13 (open
arrowhead)-kDa glycopeptides, potential N-glycosylation
sites (asterisks), the estimated C termini of the two peptides (vertical bars), and the two similar octapeptide sequences (boxed). B, diagram showing the location of the
tryptic glycopeptides in relation to the carbonic anhydrase- and
fibronectin type III-like domains of phosphacan/RPTP/
. The numbers indicate the amino acid residues corresponding to the
N termini and the estimated C termini of the peptides and to the domain
boundaries.
In view of the fact that two peptides were observed to bind to the neural cell adhesion molecules and tenascin and that both peptides contained potential N-glycosylation sites (at Asn-232 and Asn-381), we considered the possibility that oligosaccharides might be involved in these interactions. Moreover, there was a blank at Asn-381 in our amino acid sequence of the 13-kDa tryptic peptide (see above), indicating that this glycosylation site was utilized. Following peptide N-glycosidase treatment of phosphacan, the electrophoretic mobility of the tryptic peptides increased and appeared as a probably overlapping broad band of the two peptides at approximately 11 kDa (Fig. 1, E-H). Based on this result and the residue weights, it can be estimated that the C termini of the two peptides are probably at Lys-303 and Lys-456. Although the decrease in molecular size following deglycosylation of the larger glycopeptide is greater than can be accounted for by the size of a complex oligosaccharide, this result is nevertheless consistent with the anomalously slow electrophoretic migration of certain glycopeptides and glycoproteins, which varies with oligosaccharide and protein structure, due to decreased binding of SDS (Segrest and Jackson, 1972; Leach et al., 1980).
Peptide N-glycosidase treatment of phosphacan reduced its binding to the neural cell adhesion molecules and tenascin by 65-75% but had little or no effect on the binding of neurocan (Fig. 3), which is a member of the aggrecan-versican family of hyaluronic acid-binding proteoglycans and has no known structural similarity to phosphacan (Rauch et al., 1992). These results are in good agreement with our previous demonstration that, in distinction to phosphacan (Milev et al., 1994), the interactions of neurocan with neural cell adhesion molecules are to a large extent mediated by its chondroitin sulfate chains (Friedlander et al., 1994). Similar effects of N-deglycosylation were observed when binding was measured in buffer containing 150 mM rather than 50 mM NaCl, although as demonstrated previously the percent bound is somewhat lower in 150 mM NaCl (Milev et al., 1994). The residual phosphacan binding after peptide N-glycosidase treatment probably reflects incomplete deglycosylation of the unreduced protein under nondenaturing conditions in which the oligosaccharides may not be completely accessible to the enzyme (Tarentino et al., 1985; Hirani et al., 1987; Nuck et al., 1990), and this is apparent in the small amounts of the 17- and 13-kDa glycopeptides that are obtained from glycosidase-treated phosphacan (Fig. 1, lanes F and H). As expected, there were only traces of radioactivity bound to Ng-CAM and N-CAM using tryptic peptides prepared from peptide N-glycosidase-treated phosphacan (data not shown). Because there are developmental changes in the glycosylation of phosphacan (Rauch et al., 1991), we also tested binding and the effects of N-deglycosylation using proteoglycan isolated from both 7-day postnatal and adult brain, but no age-related differences were found ( Fig. 1and Fig. 3).
Figure 3:
Effects of N-deglycosylation on
the binding of phosphacan (from 7-day postnatal and adult brain) and
neurocan to Ng-CAM/L1, N-CAM, and tenascin. Ng-CAM and N-CAM were
coated in removable wells at a concentration of 2 µg/ml and
tenascin at a concentration of 20 µg/ml. Labeled proteins were used
at 90,000 cpm/well. Solid bars, untreated proteoglycan; open bars, control incubation (6 h at 37 °C in digestion
buffer); shaded bars, treatment with peptide N-glycosidase (PNGase); hatched bars,
treatment with peptide N-glycosidase/Endo F. The percent bound
represents specific binding (total counts/min bound minus counts/min
bound to wells coated with BSA), and values are the averages of two to
four determinations ± S.E.
Electrophoresis of glycosidase-treated phosphacan showed no evidence of protease activity in the enzyme preparation, and control experiments showed that residual glycosidase present in the diluted proteoglycan used for the binding assay did not affect binding by acting on the CAM or tenascin during the binding incubation period. In fact, treatment of Ng-CAM with peptide N-glycosidase increased the binding of phosphacan by nearly 100%, but peptide N-glycosidase treatment of N-CAM and tenascin had no effect on phosphacan binding (data not shown). Moreover, glycosidase treatment of Ng-CAM had no detectable effect on the binding of phosphacan tryptic glycopeptides, suggesting that N-linked oligosaccharides on Ng-CAM may modulate its interactions with phosphacan by affecting Ng-CAM conformation or through steric factors.
Monosaccharide analyses of the two tryptic
glycopeptides showed in both cases the presence of glucosamine,
mannose, galactose, and sialic acid in molar ratios characteristic of
triantennary oligosaccharides (Table 1). A large portion of the
oligosaccharides on the 17-kDa glycopeptide also contain fucose. These
results are consistent with the finding that treatment of phosphacan
with Endo H, which releases high-mannose and hybrid oligosaccharides,
had no effect on its binding to Ng-CAM, N-CAM, or tenascin (data not
shown). We have previously found that phosphacan contains
oligosaccharides with 3-sulfated residues that are recognized by the
HNK-1 monoclonal antibody (Rauch et al., 1991), as well as
Lewis oligosaccharides (Allendoerfer et al., 1995)
that are not present on other soluble chondroitin sulfate proteoglycans
of rat brain. (
)However, staining of immunoblots
demonstrated that neither of the tryptic glycopeptides that bind to
neural cell adhesion molecules and tenascin show HNK-1 or Lewis
reactivity, indicating that these structures are not involved in
the interactions we have described.
The only amino acid sequence similarity between the two tryptic glycopeptides resides in the octapeptides ILQNLLPN (beginning at residue 215) and ILNNLIPN (beginning at residue 374), which have 75% identity (Fig. 2A). A synthetic peptide containing the ILNNLIPN sequence and corresponding to a lysine followed by the first 21 amino acids of the 13-kDa glycopeptide produced up to 60% inhibition of phosphacan binding to Ng-CAM. However, there was even greater inhibition by a scrambled control peptide having the same amino acid composition, and binding inhibition studies using the first or last ten amino acids of this peptide sequence as well as a number of unrelated peptides also failed to support the involvement of these octapeptide sequences in phosphacan binding (Fig. 4). The highly variable inhibitory effects of the tested peptides probably reflects their ability to interact nonspecifically with the oligosaccharides involved in phosphacan binding.
Figure 4:
Effects of synthetic peptides on binding
of I-phosphacan to Ng-CAM. The peptides used were:
KHEFLTDGYQDLGAILNNLIPN (
, N terminus of 13-kDa tryptic
glycopeptide, abbreviated as KHE); NGFYLHNALKPDTIGLIELDNQ (
,
scrambled version of KHE peptide); KHEFLTDGYQD (
, N-terminal
half of KHE peptide); LGAILNNLIPN (
, C-terminal half of KHE
peptide); PFHSSDIQRDELAPSGTGVSRE (
, from amyloid precursor-like
protein 1); YGGFMTSEKSQTPLVTLFKNAIIKNAHKKGQ (
,
-endorphin);
GGNRNNFDTEEYC (
, from the amyloid precursor protein); and
CPKVNPHGSGPEEKRHR (
, from glypican). Points represent the averages
of duplicate determinations.
Although there has been considerable interest in the
function of glycans in molecular recognition, protein sorting, and
developmental processes, the dramatic decrease in binding of
phosphacan/RPTP/
to Ng-CAM/L1, N-CAM, and tenascin following
removal of N-linked oligosaccharides represents one of the
still relatively few instances in which they have been demonstrated to
play a role in protein interactions in vertebrate tissues. Because
glycosylation is tissue- and cell type-specific (Parekh et
al., 1987, 1989), we chose to utilize for our studies phosphacan
purified from brain, since recombinant forms may not address the
function of the native proteoglycan, and this is presumably the reason
why carbonic anhydrase and fibronectin type III domains of
phosphacan/RPTP
/
expressed as Fc fusion proteins in COS7 or
293 cells were not capable of binding to Ng-CAM, N-CAM, or tenascin
(Peles et al., 1995). The lack of binding of a recombinant
protein containing the phosphacan/RPTP
/
amino acid sequence
up to Leu-415 provides additional evidence for the role of specific
oligosaccharides in the binding process.
Our results suggest that
the neural cell adhesion molecules and tenascin, all of which are
involved in cell interactions, contain a lectin sequence that binds the
phosphacan/RPTP/
oligosaccharides, and like certain other
lectins, the binding of phosphacan by tenascin requires the presence of
divalent cations.
The phosphacan binding site may be
located in the Ig-like domains of Ng-CAM and N-CAM, since it has
previously been reported that the fourth Ig-like domain of N-CAM
contains a recognition site for high-mannose oligosaccharides on L1
(Horstkorte et al., 1993) and that a soluble immunoglobulin
fusion construct of CD22, a B cell-specific receptor of the
Ig-superfamily, binds to the trisaccharide
Sia(
2-6)Gal(
1-4)GlcNAc that is present in N- and O-linked oligosaccharides (Powell et
al., 1995).
Other oligosaccharides may also participate in the
binding process in addition to those on the two identified tryptic
glycopeptides. The affinity of phosphacan/RPTP/
for neural
cell adhesion molecules and tenascin is several orders of magnitude
greater than that for most lectin interactions with monovalent
oligosaccharide ligands, which are usually in the micromolar range (Lee
and Lee, 1994), and is therefore consistent with multiple interactions.
A more detailed structural characterization of the
phosphacan/RPTP
/
glycans is clearly of interest, both because
this information may be useful for the design of agents that affect
interactions of a receptor-type protein-tyrosine phosphatase with its
ligands, and insofar as the asparagine-linked oligosaccharides involved
in the binding of phosphacan to neural cell adhesion molecules and
tenascin are also likely to mediate its potent inhibitory effects on
cell adhesion and neurite outgrowth that we reported previously (Milev et al., 1994).