(Received for publication, June 20, 1995)
From the
The glycan core structures of the glycosyl-phosphatidylinositol
(GPI) anchors on porcine and human renal membrane dipeptidase (EC
3.4.13.19) were determined following deamination and reduction by a
combination of liquid chromatography, exoglycosidase digestions, and
methylation analysis. The glycan core was found to exhibit
microheterogeneity with three structures observed for the porcine GPI
anchor: Man1-2Man
1-6Man
1-4GlcN (29% of
the total population),
Man
1-2Man
1-6(GalNAc
1-4)Man
1-4GlcN
(33%), and
Man
1-2Man
1-6(Gal
1-3GalNAc
1-4)Man
1-4GlcN
(38%). The same glycan core structures were also found in the human
anchor but in slightly different proportions (25, 52, and 17%,
respectively). Additionally, a small amount (6%) of the second
structure with an extra mannose
(1-2)-linked to the
non-reducing terminal mannose was also observed in the human membrane
dipeptidase GPI anchor. A small proportion (maximally 9%) of the
porcine GPI anchor structures was found to contain sialic acid,
probably linked to the GalNAc residue. The porcine GPI anchor was found
to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion
electrospray-mass spectrometry revealed the presence of exclusively
diacyl-phosphatidylinositol (predominantly
distearoyl-phosphatidylinositol with a minor amount of
stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane
dipeptidase anchor. Porcine membrane dipeptidase was digested with
trypsin and the C-terminal peptide attached to the GPI anchor isolated
by removal of the other tryptic peptides on anhydrotrypsin-Sepharose.
The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser,
thereby identifying the site of attachment of the GPI anchor as
Ser
. This work represents a comprehensive study of the
GPI anchor structure of porcine membrane dipeptidase and the first
interspecies comparison of mammalian GPI anchor structures on the same
protein.
Glycosyl-phosphatidylinositol (GPI) ()membrane
anchors are present in organisms at most stages of eukaryotic
evolution, including protozoa, yeast, slime molds, invertebrates, and
vertebrates, and are found on a diverse range of proteins. They are
primarily responsible for the anchoring of cell-surface proteins in the
plasma membrane and may be considered as an alternative to the
hydrophobic transmembrane polypeptide anchor of integral membrane
proteins. Many other functions have been proposed for GPI anchors,
including roles in intracellular sorting, transmembrane signaling, and
the novel endocytic process of potocytosis. A GPI anchor might also
allow the protein to associate in membrane microdomains or be
selectively released from the cell-surface by phospholipases. These
functions, as well as the structure, biosynthesis, and distribution of
GPI anchors have been extensively reviewed (Ferguson and Williams,
1988; Cross, 1990; Thomas et al., 1990; Ferguson, 1991;
Ferguson 1992a; Englund, 1993; McConville and Ferguson, 1993).
Although over 100 examples of GPI-anchored proteins have been described very few GPI anchor structures have been characterized in detail. To date partial or complete structures have been described in a variety of protozoal proteins, including Trypanosoma brucei variant surface glycoprotein (VSG) (Ferguson et al., 1988) and procyclic acidic repetitive protein (PARP) (Field et al., 1991; Ferguson et al., 1993), Leishmania major promastigote surface protease (Schneider et al., 1990), Trypanosoma cruzi 1G7 antigen (Güther et al., 1992), and Tc85 glycoprotein (Couto et al. 1993). They have also been characterized in yeast glycoproteins (Fankhauser et al., 1993), Dictyostelium discoideum prespore-specific antigen (Haynes et al., 1993), and Torpedo electric organ acetylcholinesterase (Mehlert et al., 1993). To date eight GPI structures on mammalian proteins have been characterized: rat brain Thy-1 antigen (Homans et al., 1988), human erythrocyte acetylcholinesterase (Roberts et al., 1988a; Deeg et al., 1992), hamster brain scrapie prion protein (Stahl et al., 1992), bovine liver 5`-nucleotidase (Taguchi et al., 1994), human placental alkaline phosphatase (Redman et al., 1994), human urine CD59 (Nakano et al., 1994), mouse skeletal muscle neural cell adhesion molecule (NCAM) (Mukasa et al., 1995), and human spleen CD52 (Treumann et al., 1995) and these are detailed in Fig. 1.
Figure 1:
Structures of known mammalian
GPI anchors. The conserved core region contains an extra ethanolamine
phosphate (EtNP) in all mammalian structures. Various side
chain modifications of carbohydrate, additional EtNP, and/or palmitate (R-R
) are found as indicated
(see text for references except human and porcine membrane dipeptidase;
this study). In some proteins certain residues may only be present in a
proportion of GPI anchors, and this is indicated by ±. OH
indicates that no modification is thought to be present. R
= lipid moiety present. n.d. = not determined.
From these studies it is apparent that the GPI anchor
consists of a highly conserved core structure of
ethanolamine-PO-6Man
1-2Man
1-6Man
1-4GlcNH
1-6myo-inositol-1-PO
-lipid.
Often attached to this conserved core are variable side chains which
may be protein-, tissue-, and/or species-specific (McConville and
Ferguson, 1993). Examples include an
-galactose branch on some T. brucei VSG molecules and additional
-mannose
residue(s) on a number of structures (including yeast, slime mold and
mammalian glycoproteins, and T. cruzi 1G7 antigen). A single
sialic acid (N-acetylneuraminic acid, NANA) residue is present
on a proportion (30%) of the scrapie prion protein anchors (Stahl et al., 1992) while an average of five sialic acid residues
are found on the T. brucei PARP anchor (Ferguson et
al., 1993). In contrast to lower eukaryotic GPI anchor structures,
all metazoan GPI structures studied to date contain at least one
additional ethanolamine phosphate residue, although the exact
position(s) and linkages have only been determined in rat brain Thy-1
and human erythrocyte acetylcholinesterase (Homans et al.,
1988; Roberts et al., 1988a; Deeg et al., 1992). The
function, if any, of side chain modifications in general remains
obscure, although the
-galactose branch in VSG has been suggested
to be involved in the dense packing of the protective surface coat of
the trypanosome (Homans et al., 1989).
From the lipid moieties that have so far been determined it appears that this part of the anchor structure can be quite variable. They range from ceramide in most yeast glycoproteins (Fankhauser et al., 1993) and slime mold pre-spore antigen (Haynes et al., 1993), to sn-1-alkyl-2-acylglycerols in L. major promastigote surface protease (Schneider et al., 1990), T. cruzi 1G7 antigen (Heise et al., 1995), human erythrocyte acetylcholinesterase (Roberts et al., 1988a, 1988b), human placental alkaline phosphatase (Redman et al., 1994), and several other mammalian GPI-anchored proteins (reviewed in McConville and Ferguson, 1993), sn-1,2-diacylglycerols in T. brucei VSG (Ferguson et al., 1985), Torpedo acetylcholinesterase (Butikofer et al., 1990) and human CD52 (Treumann et al., 1995), and an unusual sn-1-acyl-2-lyso-glycerol in T. brucei PARP (Field et al., 1991). In addition, several GPI anchors contain an additional fatty acid (palmitate) in ester linkage to the myo-inositol ring. Examples include human erythrocyte acetylcholinesterase (Roberts et al., 1988b), T. brucei PARP (Field et al., 1991; Ferguson, 1992c) and one form of human CD52 (Treumann et al., 1995). The latter study showed that the modification is at the 2-position of the myo-inositol ring.
The mRNAs of known GPI-anchored proteins encode an N-terminal
signal sequence, to direct the protein to the endoplasmic reticulum,
and a C-terminal GPI attachment signal sequence. This sequence is
believed to hold the nascent protein in the membrane prior to anchor
addition and is cleaved post-translationally with the concomitant
addition of the GPI anchor. The anchor attachment site () may be
one of six amino acid residues, all of which have small side chains
(Ala, Asn, Asp, Cys, Gly, or Ser) (Moran et al., 1991; Gerber et al., 1992). In addition, the residue at the
+ 2
position is restricted to Ala, Gly, or Ser (Gerber et al.,
1992).
Membrane dipeptidase (MDP, dehydropeptidase-I, renal
dipeptidase, microsomal dipeptidase, EC 3.4.13.19) is a
zinc-metalloenzyme found predominantly in the brush-border membrane of
the kidney and also in the lung (for review see Keynan et al.,
1995). The enzyme is capable of hydrolyzing a wide variety of
dipeptides (Campbell, 1970; Armstrong et al., 1974) may have a
role in the metabolism of glutathione and leukotriene D (Kozak and Tate, 1982; Campbell et al., 1990) and is the
only known mammalian enzyme to exhibit
-lactamase activity (Kropp et al., 1982). Porcine MDP was the first mammalian peptidase
that was found to be anchored via GPI (Hooper et al., 1987)
and subsequently human MDP was also shown to be GPI-anchored (Hooper
and Turner, 1988). The GPI anchor on both porcine and human MDP has
been extensively characterized in terms of its hydrolysis by bacterial
phosphatidylinositol-specific phospholipase C (PI-PLC), and a serum
phospholipase D (Littlewood et al., 1989; Hooper and Turner,
1989; Hooper et al., 1990; Brewis et al., 1994) and
polyclonal antisera have been generated against the cross-reacting
determinant of both proteins (Hooper et al., 1991; Broomfield
and Hooper, 1993).
In the present study we have continued our molecular characterization of the GPI anchors on porcine and human MDP. The complete structure of the porcine MDP GPI anchor has been determined, including the nature of the lipid species present, the extent of sialylation, and the site of GPI anchor attachment in the polypeptide chain. In addition, we have compared the GPI anchor glycan core structures of porcine and human MDP.
To measure the extent of desialylation due
to the mildly acidic conditions of the aqueous HF dephosphorylation
step, NaBH
-reduced 3`-sialyl-lactitol
(NANA
2-3Gal
1-4[1-
H]glucitol)
and 6`-sialyl-lactitol
(NANA
2-6Gal
1-4[1-
H]glucitol)
standards (10 nmol of each) were subjected to aqueous HF treatment and N-acetylation as described above. The extent of desialylation
was measured by separating the neutral desialylated product
(Gal
1-4[1-
H]glucitol) from the
starting material by HVE. The neutral and acidic glycans were
quantitatively eluted from the electrophoretogram and taken for
scintillation counting. From this data the extent of desialylation was
found to be 31 and 52% for 3`-sialyl-lactitol and 6`-sialyl-lactitol,
respectively.
Figure 2: Liquid chromatography of the neutral glycans of porcine membrane dipeptidase. Radiolabeled neutral glycans were separated on Dionex HPLC into two peaks of 2.5 and 3.0 Du (panel a). Each peak (2.5 Du peak, panel b; 3.0 Du peak, panel c) was further chromatographed by Bio-Gel P4 gel-filtration.
A preliminary set of exoglycosidase digestions on
the unknown porcine neutral glycan (structure B) were performed and
assessed by Dionex HPLC and/or Bio-Gel P4 gel-filtration (data not
shown). Digestions with jack bean -hexosaminidase, jack bean
-galactosidase, and coffee bean
-galactosidase were all
negative. However, bovine testes
-galoactosidase successfully
digested all of structure B to structure C. Resistance to jack bean
-galactosidase and sensitivity to bovine testes
-galoactosidase is consistent with the presence of a non-reducing
terminal
Gal residue linked 1-3 to a subterminal residue (Li et al., 1975; Distler and Jourdain, 1978). Thus neutral glycan
structure B appears to differ from structure C by the presence of a
non-reducing terminal 1-3-linked
Gal residue. The
provisional assignment of the neutral glycan structures from their
chromatographic properties greatly assisted in designing the minimum
number of exoglycosidase digestions needed to sequence them. An
authentic standard of
Man
1-2Man
1-6Man(GalNAc
1-4)Man
1-4AHM
isolated from rat brain Thy-1 (Homans et al., 1988) was used
as a control throughout. Two series of exoglycosidase digestions were
performed on the porcine neutral glycans, and the products were
analyzed by HPTLC (Fig. 3, a and b). Identical
results were obtained for the human neutral glycan structures B, C, and
D (data not shown), and the results for the human neutral glycan
structure A are shown in Fig. 3c. The digestions shown
in Fig. 3a define the position of the
Gal
1-3GalNAc
1 and GalNAc
1 branches of structures B
and C to the
Man residue adjacent to the AHM.
Figure 3:
Thin layer chromatography analysis of the
exoglycosidase digestions of the isolated GPI neutral glycans of
membrane dipeptidase. The porcine neutral glycan structures B, C, and D (panels a and b) and the human neutral glycan
structure A (panel c) were subjected to the series of
exoglycosidase digestions indicated. The number along side each
intermediate structure corresponds to the lane of the HPTLC plate shown
below. The abbreviations used are: A, 2,5-anhydromannitol; G, Gal; GN,
GalNAc, and M,
Man. The standard used as a control was authentic
Man
1-2Man
1-6(GalNAc
1-4)Man
1-4AHM
isolated from rat brain Thy-1. Each lane contains the equivalent of
5000 cpm of material. Tritium-labeled reduced
-glucose oligomer
standards (Dex, 10,000 cpm) were also chromatographed on the HPTLC
plate. Fluorography was performed at -70 °C for 1 week. ASAM, A. saitoi
-mannosidase; BTBG,
bovine testes
-galactosidase; JBAM, jack bean
-mannosidase; JBBH, jack bean
-hexosaminidase;
All of the
digestions were successful except that the first set of jack bean
-mannosidase digestions for each of the structures in Fig. 3a achieved only partial (about 60-80%)
digestion. The known standard was also only partially digested, and
therefore it was concluded that each of the samples was homogeneous and
that the jack bean
-mannosidase digestion in this step was
incomplete. The incomplete jack bean
-mannosidase digestions were
most likely due to steric hindrance from the
GalNAc residue. Each
of the samples were completely digested in the second jack bean
-mannosidase digestion which would not have been the case if there
were other structures present. The use of Man
1-2Man-specific A. saitoi
-mannosidase in Fig. 3, b and c, was important to show that the non-reducing
terminal Man residue in structures B, C, and D was linked
1-2 to the subterminal Man residue and that both the
non-reducing terminal
Man residue and the subterminal
Man
residue of neutral glycan structure A were similarly 1-2-linked.
In addition to the assessment of the exoglycosidase digestions by
HPTLC, all of the digestions performed on porcine structure D were also
assessed by Dionex HPLC and Bio-Gel P4 gel-filtration chromatography.
The APAM digestion product (Fig. 3b) eluted at 2.1 Du
and 3.2 Gu and the jack bean -mannosidase digestion products (Fig. 3, a and b) eluted at 1.0 Du and 1.7 Gu.
These chromatographic properties are consistent with
Man
1-6Man
1-4AHM and AHM, respectively (Ferguson,
1992b). All of the exoglycosidase digestions performed in Fig. 3b on the porcine structures B and C, to the point
where the structure was the same as porcine structure D, were also
assessed by Dionex HPLC and Bio-Gel P4 gel-filtration chromatography,
and these results were also consistent with the intermediate structures
presented. Identical results were also obtained for the human
structures B, C, and D. The exoglycosidase digestions of human
structure A presented in Fig. 3c were also assessed by
Dionex HPLC and Bio-Gel P4 gel-filtration chromatography. The jack bean
-hexosaminidase digestion product eluted at 3.0 Du and 5.1 Gu, the A. saitoi
-mannosidase digestion product eluted
at 2.1 Du and 3.2 Gu, and the jack bean
-mannosidase digestion
product eluted at 1.0 Du and 1.7 Gu. These results are consistent with
Man
1-2Man
1-2Man
1-6Man
1-4AHM,
Man
1-6Man
1-4AHM, and AHM, respectively (Ferguson,
1992b).
Methylation analysis of the total porcine neutral glycan
fraction (Table 2) is consistent with the exoglycosidase results
described above. The presence of 4,6-di-O-substituted Man
defines the linkage position of the GalNAc residue to the 4-position of
the Man residue adjacent to the AHM. The presence of
terminal-GalNAc is consistent with the presence of structure C and the
presence of terminal-Gal and 3-O-substituted GalNAc confirms
the presence of the Gal
1-3GalNAc branch proposed for
structure B. Methylation analysis of the N-acetylated glycan
core sample (Table 2) showed, in addition, the presence of
4-O-substituted GlcNAc (in place of the
4-O-substituted AHM) and 6-O-substituted myo-inositol. These results further define the original GPI
structures as containing GlcN1-6-myo-inositol termini.
Figure 4:
Ion-exchange chromatography of an N-acetylated glycan core fraction of porcine membrane
dipeptidase. GPI glycans were prepared from porcine MDP by deamination,
reduction, dephosphorylation, and N-acetylation. The glycans
(containing a radioactive tracer) were then subjected to ion-exchange
chromatography on a QAE-Sephadex A25 (OH) column.
Three radioactive peaks (labeled 1-3, solid
line) were resolved, eluted, and analyzed for monosaccharide
composition by GC-MS. Also presented is the ammonium acetate gradient
applied to the column (dashed line). See text for
details.
Figure 5:
ES-MS analysis of the PI fraction of
porcine membrane dipeptidase. The PI fraction of porcine MDP was
analyzed by negative ion ES-MS (panel a). The pseudomolecular
ions of PI species were detected in the ES-MS-MS parent ion spectrum (panel b) of the PI-specific fragment ion
[inositol-1,2-cyclic phosphate] (m/z 241). The ES-MS-MS daughter ion spectrum of the m/z 865
parent ion (panel c) shows the presence of the m/z 241 [inositol-1,2-cyclic phosphate]
fragment ion and an m/z 283
[CH
(CH
)
COO]
carboxylate fragment ion.
Figure 6:
Comparison of the deduced C-terminal amino
acid sequence of porcine membrane dipeptidase with the cDNAderived
amino acid sequences of porcine and human membrane dipeptidase. a, determined C-terminal peptide sequence of porcine
dipeptidase. b, porcine dipeptidase cDNA-derived protein
sequence (Rached et al., 1990). c, human dipeptidase
cDNA-derived protein sequence (Adachi et al., 1990a). S = site of GPI anchor attachment. = site
of cleavage by trypsin. The porcine and human cDNA sequences are
aligned to give maximum homology between them, hence the two gaps
(-) in the porcine sequence. Numbering refers only to the porcine
sequence.
A combination of Dionex HPLC and Bio-Gel P4 gel-filtration (Fig. 2), exoglycosidase digestions (Fig. 3), and
methylation analyses (Table 2) produced a complete qualitative
and quantitative analysis of the glycan cores of MDP isolated from both
porcine and human kidney (Table 1). All of the structures
identified contained the conserved GPI core sequence found throughout
eukaryotic evolution of
Man1-2Man
1-6Man
1-4GlcN (McConville and
Ferguson, 1993). The carbohydrate side chains found in both MDP samples
included GalNAc and Gal
1-3GalNAc, both linked
1-4
to the conserved core (see Fig. 1). In human MDP one additional
minor structure, containing an extra
Man residue, as well as a
GalNAc side chain, was also observed. This is the first trans-species
comparison of the GPI anchor structures of the same protein expressed
in the same tissue. It is noteworthy that the porcine and human anchor
preparations display identical structures in quite similar molar
ratios, thus suggesting that side chain modifications to GPI anchors
are not species-specific but rather protein- and/or tissue-specific.
Interestingly, the side chain modifications (an extra
Man residue
and a GalNAc) to the GPI anchor of human urine CD59 (Nakano et
al., 1994), which is probably derived from the epithelial cells
lining the kidney tubules, are similar to those found on human MDP (see Fig. 1).
Microheterogeneity of glycan core structures has
also been commonly observed with other mammalian GPI structures
although none is found either in human erythrocyte acetylcholinesterase
(Roberts et al., 1988a) or human placental alkaline
phosphatase (Redman et al., 1994). The presence of
GalNAc-linked 1-4 to the conserved GPI core has been
previously described in rat brain Thy-1 (Homans et al., 1988), Torpedo acetylcholinesterase (Mehlert et al., 1993),
mouse skeletal muscle NCAM (Mukasa et al., 1995) and human
urine CD59 (Nakano et al., 1994), and HexNAc has been observed
in hamster brain scrapie prion protein (Stahl et al., 1992)
and bovine liver 5`-nucleotidase (Taguchi et al., 1994). The
presence of the Gal
1-3GalNAc side chain has not been
previously noted in GPI anchor structures. However, it is reminiscent
of the partial structure described for the hamster brain scrapie prion
protein GPI anchor of Hex-HexNAc and NANA-Hex-HexNAc side chains (Stahl et al., 1992). The Gal
1-3GalNAc motif is also
common in glycoprotein and mucin O-linked oligosaccharides
(Carlstedt et al., 1985; Van Halbeek et al., 1988;
Hokke et al., 1994), and it is possible that the same
1-3 galactosyltransferase is used to modify both O-linked GalNAc residues and GPI anchor GalNAc residues.
The possibility that some of the GPI glycans were originally
sialylated was investigated by releasing the GPI anchor of porcine MDP
by aqueous HF dephosphorylation under conditions that retain half to
two-thirds of the sialic acid residues, depending on the linkage. The
low yield of sialylated GPI glycans obtained after anion-exchange
chromatography suggested that a maximum of 9% of the original GPI
anchors contained sialic acid (presuming 2-6 linkage and no
polysialylation). The low abundance of sialylated species, and their
inherent lability to the aqueous HF conditions necessary to prepare
them, prevented a detailed structural characterization. However, the
composition of the sialylated fraction suggests that the presence of
GalNAc is a prerequisite for sialylation whereas only a small fraction
of the sialylated species contained Gal. This leads us to tentatively
suggest that in the case of the porcine MDP GPI anchor most, if not
all, of the sialic acid is attached to the side chain GalNAc residue.
The presence of sialic acid in GPI anchors has been reported only
twice. In one case, that of T. brucei PARP, an average of 5
sialic acid residues/anchor were found linked
2-3 to the
terminal
Gal residues of branched polylactosamine side chain
structures (Ferguson et al., 1993). In the other example, that
of hamster brain scrapie prion protein, about 30% of the anchors were
found to be monosialylated with the sialic acid attached to a
Hex-HexNAc side chain, predominantly in the form NANA-Hex-HexNAc (Stahl et al., 1992). Thus, the possibility exists that, like O-linked oligosaccharides and ganglioside glycans, sialic acid
can be linked to both Gal and GalNAc residues in GPI anchor side
chains.
The porcine MDP was shown to contain 2.5 mol of ethanolamine/mol of GPI anchor indicating that, in addition to the ethanolamine phosphate bridge, there is an extra ethanolamine phosphate moiety in the GPI anchor and, probably in half the structures, a third ethanolamine phosphate residue is present. The presence of at least one extra ethanolamine phosphate in the GPI anchors of metazoan (but significantly not unicellular) eukaryotes is well documented (McConville and Ferguson, 1993). Deeg et al.(1992) first precisely analyzed the presence of two additional ethanolamine phosphates in human erythrocyte acetylcholinesterase in 10-20% of the structures. Two additional ethanolamine phosphates are also thought to occur in a proportion of the GPI anchors of bovine liver 5`-nucleotidase (only 3.5% of total population) (Taguchi et al., 1994), Torpedo acetylcholinesterase (up to 30% by compositional analysis) (Mehlert et al., 1993), human placental alkaline phosphatase (up to 40% by compositional analysis) (Redman et al., 1994), and human CD52 (40% of the total population) (Treumann et al., 1995). The presence of two additional ethanolamine phosphate residues was not found in rat brain Thy-1 nor hamster brain scrapie prion protein suggesting that this is not a ubiquitous feature of all metazoan GPI structures (Homans et al., 1988; Stahl et al., 1992). The presence of this third ethanolamine phosphate in about half of the porcine MDP anchors would cause microheterogeneity of charge, in addition to the microheterogeneity of the carbohydrate residues.
In general, the GPI PI moieties are substantially different from the cellular pool of PI phospholipids (McConville and Ferguson, 1993). For example, several of the mammalian GPI anchors contain exclusively alkylacyl-PIs (Roberts et al., 1988b; Walter et al., 1990; Redman et al., 1994) (see Fig. 1) as opposed to sn-1-stearoyl-2-arachidonoyl-PI that is the predominant cellular PI species in these organisms (Michell, 1975; Kerwin et al., 1994). In the case of porcine MDP, the predominant PI moiety is a diacyl-PI (distearoyl-PI). Although diacyl-PI moieties are quite common in non-mammalian GPI anchors (e.g. in Torpedo acetylcholinesterase (Bütikofer et al., 1990), T. brucei VSG (Ferguson et al., 1985), Saccharomyces cerevisiae gp125 (Fankhauser et al., 1993), and human CD52 (Truemann et al., 1995)), the presence of predominantly distearoyl-PI has only been reported previously for one of the two forms of human CD52 (Truemann et al., 1995). The only other example of a GPI anchor diacyl-PI that contains predominantly one type of acyl chain is that of T. brucei VSG. The dimyristoyl-PI moiety of the VSG anchor is produced by a process of fatty acid remodeling (Masterson et al., 1990), whereby the original heterogeneity in the PI moiety (Doering et al., 1994) is removed by sequentially replacing the sn-2 and sn-1 fatty acids with myristate. While most mammalian GPI intermediates and precursors contain alkylacyl-PIs, some contain diacyl-PIs (Puoti and Conzelmann, 1993). Thus, it is possible that some kind of analogous fatty acid remodeling may occur on the diacyl-PI containing GPI intermediates in the cells producing CD52 and porcine MDP. Alternatively, these cells may simply select for distearoyl-PI as the precursor for the GPI intermediates that will be transferred to these proteins.
The results of the trypsin digestion
of porcine MDP and isolation of the C-terminal peptide on AHT-Sepharose
identified the site of GPI anchor attachment as Ser. Thus
the mature protein lacks the predominantly hydrophobic C-terminal
sequence of 25 residues predicted from the cDNA (Rached et
al., 1990). The Ser anchor attachment site (
) has a pair of
Ala residues immediately C-terminal to it. This is consistent with the
proposals of Gerber et al.(1992), who have found that the next
but one residue C-terminal to the site of anchor attachment (
+ 2 position) is generally restricted just to Ala, Gly, or Ser.
Also displayed in Fig. 6is the cDNA-derived protein sequence
for human MDP and the previously determined GPI anchor attachment site
of Ser
(Adachi et al., 1990b). The
human and porcine proteins are 80% homologous and, from sequence
alignment studies, it appears that in the porcine MDP either
Ser
or Ser
has been mutationally deleted.
However, it is impossible to predict from the cDNA codon information
which Ser is absent. Whatever the case both the enzymes utilize Ser in
the same part of the protein sequence for GPI anchor attachment and
have Ala at the
+ 2 position.
In conclusion, we have
determined the glycan core structures of the GPI anchors on porcine and
human MDP, representing the first interspecies comparison of mammalian
GPI anchor structures on the same protein. The glycan core structures
were remarkably similar, with three major structures observed in
approximately equal amounts:
Man1-2Man
1-6Man
1-4GlcN,
Man
1-2Man
1-6(GalNAc
1-4)Man
1-4GlcN,
and
Man
1-2Man
1-6(Gal
1-3GalNAc
1-4)Man
1-4GlcN.
Also the GPI anchor on porcine MDP was found to be anchored to the
protein at Ser
, to contain 2.5 mol of ethanolamine/mole
of anchor, and almost exclusively distearoyl-PI. In addition, a small
proportion of the porcine GPI structures were shown to contain sialic
acid residues, probably attached to GalNAc.