GPI proteins in chickens
GPI proteins in Drosophila
GPI in prion proteins
Human GPI proteins: protectin (CD59) and homologous restriction factor (HRF, C8bp, MIP)
GPI proteins in yeast (14 in whole genome
Prion oligosaccharide abstracts
Prion proteins from all species sequenced so far (from human to chicken) contain a C-terminal GPI-anchor, a post-translational modification that replaces an initial hydrophobic terminus with glycosyl phophotidylinositol, serving to attach the mature protein to the outside of the cytoplasmic membrane, while allowing for later release by phospholipase. It seems likely that salmon prion will contain this modification, pushing the date of origin of this modification of prion protein back to at least 440 million years ago.
GPI-anchors are not common as a protein modification. In yeast, where all GPI proteins are known, only 15 of the 5,790 (so 1 in 400) proteins have GPI-anchors and even these have considerable homology with each other. When mammalian homologues of yeast GPI proteins are known, these have the GPI-modification as well. In such protein families, the modification has evidently been in continuous use for a billion years or more. Yeast GPI biosynthetic enzymes are likewise homologous to mammals counterparts.
GPI proteins have been found in a wide variety of eucaryotes : mammals (45 in humans), chickens (10), fish, rays, sea urchin, fruit flies (5), silk moth, ticks, grasshopper, protozoa (trypanosomes, leishmanii, paramecium), fungi, slime mold, unicellular green alga, mung bean, even herpes virus (simian surface glycoprotein), but not in bacteria, and oddly nothing reported from nematode (out of 1208 proteins).
GPI-proteins are evenly split between enzymes and binding, recognition, and transport non-catalytic proteins. This split correlates fairly strongly with whether internal tandem repeats are present (non-catalytic) or not (catalytic). A GPI-anchor unsurprisingly implies a signal peptide but by no means conversely. There is a division between O- and N-glycosylation. Identified functions are clearly appropriate to the extra-cytoplasmic location; GPI proteins are over-represented in neurons.
Human genetic disorders have been identified in five GPI proteins other than prion These include Marfan's syndrome [FBN1 gene] and Charcot-Leyden crystals [LPPL gene], alkaline phosphatase: infantile hypophosphatasia, lipoprotein lipase: chylomicronemia syndrome, and glypican-3: Simpson-Golabi-Behmel syndrome> A defect in a GPI biosynthetic enzme causes paroxysmal nocturnal hemoglobinuria.
The question arises, how ancient is the GPI-anchor in prion protein, how often and which sorts of new proteins are recruited to GPI-status, how many super-families of GPI proteins exist and to which does prion protein belong, what sort of internal structural motifs occur, where are close but non-GPIhomologues located in the cell, is prion protein more likely catalytic than structural, and finally, does analysis of GPI-proteins help identify the normal function of prion protein or suggest other candidates for prionic disease?
Brown D; Waneck GL J Am Soc Nephrol 3: 895-906 (1992)Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane.
GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase.
GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme.
At least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins:
(1) it is a strong apical targeting signal in polarized
(2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli;
(3) GPI-anchored proteins can act as activation antigens in the immune system;
(4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated;
(5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules.
Identifying Lipid Anchors:
The protein modification reactions which bind lipid molecules to proteins are important because a linked lipid moiety can be integrated into various membranes and can anchor the bound protein. All proteins linked to the glycosyl-phosphatidylinositol (GPI) molecules are thought to be anchored at the extracellular surface of the plasma membrane. PSORT recognizes GPI-anchored proteins by the knowledge that most of them are predicted to be type Ia membrane proteins with very short cytoplasmic tail (within 10 residues) and uses the result for the prediction of the localization site (plasma membrane) of the modified protein.
AXO1_CHICK Axonin-1 precursor: 1036 AA P28685
Axon-associated cell adhesion molecule (axcam) which promotes neurite outgrowth by interaction with the axcam l1 (g4) of neuritic membrane. Atached to the neuronal membrane by agpi-anchor. Belongs to the immunoglobulin superfamily. contains six c2-like domains followed by four fibronectin type iii-like domains
signal 1 23 or 25 (potential).
chain 24 1036 axonin-1.
propep ? 1036 removed in mature form.
domain 49 113 ig-like c2-type domain.
domain 143 211 ig-like c2-type domain.
domain 249 308 ig-like c2-type domain.
domain 336 397 ig-like c2-type domain.
domain 428 490 ig-like c2-type domain.
domain 518 589 ig-like c2-type domain.
domain 599 608 hinge (potential).
domain 601 607 gly/pro-rich.
domain 608 709 fibronectin type-iii.
domain 710 811 fibronectin type-iii.
domain 812 912 fibronectin type-iii.
domain 913 1009 fibronectin type-iii.
mod_res ?24 ?24 blocked.
carbohyd 71 71 potential.
carbohyd 199 199 potential.
carbohyd 456 456 potential.
carbohyd 472 472 potential.
carbohyd 493 493 potential.
carbohyd 520 520 potential.
carbohyd 770 770 potential.
carbohyd 900 900 potential.
carbohyd 914 914 potential.
EPL6_CHICK EPH-related receptor tyrosine kinase ligand 6 precursor (lerk-6)200 aa P52802
Binds to the receptor tyrosine kinases cek7, mek4 and sek.
Atached to the membrane by a gpi-anchor
Homologous to oher members of the eplg family.
signal 1 22
carbohyd 36 36
carbohyd 161 161
carbohyd 175 175
G55A_Chick Neurite inhibitor gp55a (fragment) 274 aa. q98892
Inhibits neurite outgrowth.
Atached to the membrane by a gpi-anchorsimilarity: belongs to the immunoglobulin superfamily. Contains three c2-like domains.
Belongs to the obcam subfamily, immunoglobulin fold
non_ter 1 1
domain <1 52 ig-like c2-type domain.
domain 80 138 ig-like c2-type domain.
domain 166 232 ig-like c2-type domain.
disulfid 87 131
disulfid 173 225
carbohyd 70 70
carbohyd 214 214
carbohyd 222 222
carbohyd 235 235
GLYP_CHICK Glypican-1 precursor (heparan sulfate proteoglycan core protein). P50593 550 AA.
Cell surface proteoglycan that bears heparan sulfate.
Attached to the membrane by a gpi-anchor
Belongs to the glypican family.
signal 1 20
chain 21 ? glypican-1.
propep ? 550 removed in mature form (potential).
carbohyd 76 76
carbohyd 113 113
carbohyd 382 382
carbohyd 52 52 glycosaminoglycan (potential).
carbohyd 483 483 glycosaminoglycan (potential).
carbohyd 485 485 glycosaminoglycan (potential).
carbohyd 487 487 glycosaminoglycan (potential).
LAMP_CHICK Limbic system-associated membrane protein precursor (e19s). Q98919; 338 AA.
Mediates selective neuronal growth and axon targeting. Probably serves as a recognition molecule for the formation of limbic connections
Attached to the membrane by a gpi-anchorsimilarity:
Belongs to the immunoglobulin superfamily. contains three c2-like domains. belongs to the obcam subfamily.
signal 1 28
chain 29 ? limbic system-associated membraneprotein.
propep ? 338 removed in mature form (potential).
domain 46 118 ig-like c2-type domain.
domain 146 204 ig-like c2-type domain.
domain 232 297 ig-like c2-type domain.
disulfid 53 111
disulfid 153 197
disulfid 239 290
carbohyd 40 40
carbohyd 66 66
carbohyd 136 136
carbohyd 148 148
carbohyd 279 279
carbohyd 287 287
carbohyd 300 300
carbohyd 315 315
LIPL_CHICK Lipoprotein lipase precursor (EC 184.108.40.206) (LPL) P11602 490 AA
The primary function of this lipase is the hydrolysis
of triglycerides of circulating chylomicrons and very low density
lipoproteins (vldl). The enzyme functions in the presence of
apolipoprotein c-2 on the luminal surface of vascular endothelium.
catalytic activity: triacylglycerol + h(2)o = diacylglycerol +
a fatty acid anion.
Subunit: homodimer, interact with apolipoprotein c-2.
Partial homology with other lipases (pancreatic, gastric, hepatic, lingual, lipoprotein, bacterial, etc.).
signal 1 25
chain 26 490 lipoprotein lipase.
act_site 159 159 charge relay system (by similarity).
act_site 183 183 charge relay system (by similarity).
act_site 268 268 charge relay system (by similarity).
domain 319 331 heparin-binding (potential).
disulfid 54 67
disulfid 243 266
disulfid 291 310
disulfid 302 305
disulfid 445 465
carbohyd 70 70
carbohyd 386 386
TECB_CHICK Beta-tectorin precursor. 329 AA P54097
Extracellular matrix. The n-terminal is blocked. n-glycosylated.
Tissue specificity: inner ear (basilar papilla, clear cells and lagena macula).
Contains a zp domain, which currently has been found in zp2, zp3, gp2, tgfr-3 and uromodulin.
signal 1 17
chain 18 329 beta-tectorin.
domain 91 283 zp (br> carbohyd 80 80
carbohyd 104 104
carbohyd 116 116
carbohyd 145 145
CONT_CHICK Contactin precursor (neural cell recognition molecule f11).1010 AA. P14781
Mediates cell surface interactions during nervous system development.
Belongs to the immunoglobulin superfamily. contains six c2-like domains followed by four fibronectin type iii-like domains.
signal 1 19
chain 20 ? contactin.
propep ? 1010 removed in mature form.
domain 50 113 ig-like c2-type domain.
domain 143 210 ig-like c2-type domain.
domain 247 308 ig-like c2-type domain.
domain 336 389 ig-like c2-type domain.
domain 420 482 ig-like c2-type domain.
domain 510 581 ig-like c2-type domain.
domain 593 599 gly/pro-rich.
domain 600 701 fibronectin type-iii.
domain 702 803 fibronectin type-iii.
domain 804 900 fibronectin type-iii.
domain 901 996 fibronectin type-iii.
carbohyd 200 200
carbohyd 249 249
carbohyd 329 329
carbohyd 448 448
carbohyd 464 464
carbohyd 485 485
carbohyd 512 512
carbohyd 582 582
carbohyd 924 924
CADD_CHICK T-Cadherin precursor (truncated-cadherin) (cadherin-13).712 AA. P33150
Tissue specificity: neural tissues. also found in muscles; kidney and retina.
Similarity: strong to other cadherins.
signal 1 22
propep 23 138
chain 139 693 t-cadherin.
propep 694 712 removed in mature form
repeat 139 245 cadherin 1.
repeat 246 363 cadherin 2.
repeat 364 477 cadherin 3.
repeat 478 583 cadherin 4.
repeat 583 693 cadherin 5.
carbohyd 86 86
carbohyd 382 382
carbohyd 500 500
carbohyd 530 530
carbohyd 638 638
carbohyd 671 671
lipid 693 693 gpi-anchor
Cadherins are a family of animal glycoproteins responsible for calcium-
dependent cell-cell adhesion. Cadherins preferentially interact with
themselves in a homophilic manner in connecting cells; thus acting as both
receptor and ligand. A wide number of tissue-specific forms of cadherins are
- Epithelial (E-cadherin) (also known as uvomorulin or L-CAM) (CDH1).
- Neural (N-cadherin) (CDH2).
- Placental (P-cadherin) (CDH3).
- Retinal (R-cadherin) (CDH4).
- Vascular endothelial (VE-cadherin) (CDH5).
- Kidney (K-cadherin) (CDH6).
- Cadherin-8 (CDH8).
- Osteoblast (OB-cadherin) (CDH11).
- Brain (BR-cadherin) (CDH12).
- T-cadherin (truncated cadherin) (CDH13).
- Muscle (M-cadherin) (CDH14).
- Liver-intestine (LI-cadherin).
Structurally, cadherins are built of the following domains: a signal sequence,
followed by a propeptide of about 130 residues, then an extracellular domain
of around 600 residues, then a transmembrane region, and finally a C-terminal
cytoplasmic domain of about 150 residues. The extracellular domain can be sub-
divided into five parts: there are four repeats of about 110 residues followed
by a region that contains four conserved cysteines. It is suggested that the
calcium-binding region of cadherins is located in the extracellular repeats.
Cadherins are evolutionary related to the desmogleins which are component of
intercellular desmosome junctions involved in the interaction of plaque
- Desmoglein 1 (desmosomal glycoprotein I).
- Desmoglein 2.
- Desmoglein 3 (Pemphigus vulgaris antigen).
The Drosophila fat protein is a huge protein of over 5000 amino acids that
contains 34 cadherin-like repeats in its extracellular domain.
CNTR_CHICK Ciliary neurotrophic factor receptor alpha precursor P51641 362 AA
Binds to cntf (gpa). The alpha chain provides the receptor specificity.
Subunit: heterotrimer of the alpha chain, lifr and gp130. Highly expressed in nervous system. also found
in skeletal muscle.
Belongs to the immunoglobulin superfamily. Contains one ig-like domain, contains 1 fibronectin type iii-like domain.
Belongs to the cytokine family of receptors.
signal 1 19
chain 20 334 ciliary neurotrophic factor receptor
propep 335 362 removed in mature form
domain 37 94 ig-like domain.
domain 199 300 fibronectin type-iii.
disulfid 44 87
carbohyd 58 58
carbohyd 68 68
carbohyd 140 140
carbohyd 188 188
lipid 334 334 gpi-anchor
THY1_CHICK THY-1 Membrane glycoprotein precursor (thy-1 antigen). 160 AA. Q07212;
May play a role in cell-cell or cell-ligand interactions during synaptogenesis and other events in the brain.
The n-terminal is blocked.
Developmental stage: it is detected at embryonic day 4 (ed4) in
forebrain and tectum. there is an increase in levels between ed16
and the first few days post-hatch. during ed19 to hatch a rapid
reduction in the levels is seen with a general increase in
expression in adulthood.
Tissue specificity: forebrain, cerebellum and tectum.
Belongs to the immunoglobulin superfamily, contains one v-like domain.
signal 1 19
chain 20 129 thy-1 membrane glycoprotein.
propep 130 160 removed in mature form (by similarity).
lipid 129 129 gpi-anchor (by similarity).
mod_res 20 20 pyrrolidone carboxylic acid
disulfid 28 129
disulfid 38 103
carbohyd 42 42
carbohyd 78 78
carbohyd 118 118
carbohyd 138 138
Incardona JP; Rosenberry TL Mol Biol Cell 7: 613-30 (1996)Drosophila has a single GPI-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE.
Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.
Incardona JP; Rosenberry TL Mol Biol Cell 7: 595-611 (1996)Despite advances in understanding the cell biology of GPI-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome.
Walsh FS; Doherty P Cell Biol Int Rep 15: 1151-66 (1991)A large number of glycoproteins in the central nervous system are attached to the cell membrane via covalent linkage to glycosylphosphatidylinositol (GPI). Many of them, including the drosophila fasciclin 1 as well as the mammalian glycoproteins Thy-1, TAG1, N-CAM and F11,F3, contactin are members of the immunoglobulin gene superfamily. These and other GPI-linked molecules have been implicated in key developmental events including selective axonal fasciculation and highly specific growth to and innervation of target tissues. In model systems fasciclin 1, TAG1 and N-CAM have been shown to be capable of mediating cell-cell adhesion via a homophilic binding mechanism confirming their operational classification as cell adhesion molecules (CAMs). However, of these molecules, only N-CAM has been shown to mediate a complex response (neurite outgrowth) via a homophilic binding mechanism.
Stahl N; Baldwin MA; Hecker R; Pan KM; Burlingame AL; Prusiner SB Biochemistry 31: 5043-53 (1992)The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.
Lehmann S; Harris DA J Biol Chem 270: 24589-97 (1995)Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5. Whether membrane attachment of the mutant PrP involves integration of the polypeptide into the lipid bilayer, self-association, or binding to other membrane proteins remains to be determined. Our results suggest that alterations in the membrane association of PrP may be an important feature of prion diseases.
Solomon KR; Rudd CE; Finberg RW Proc Natl Acad Sci U S A 93: 6053-8 (1996)Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1
Chen R; Udenfriend S; Prince GM; Maxwell SE; Ramalingam S; Gerber LD; Knez J; Medof ME Proc Natl Acad Sci U S A 93: 2280-4 (1996)The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane.
Coyne KE; Crisci A; Lublin DM J Biol Chem 268: 6689-93 (1993)Many membrane proteins are anchored to the cell surface through covalent attachment to a glycosyl-phosphatidylinositol (GPI) structure. The GPI anchor is added to proteins in the endoplasmic reticulum following recognition of a signal in the COOH terminus of the protein. We show that the GPI anchoring signal can be completely recreated by the synthetic polymer Ser3-Thr8-Leu14, but not Thr11-Leu14, inserted at the COOH terminus of a protein. This is consistent with previous reports that a small amino acid such as Ser, Gly, or Ala, but not Thr, is required at the GPI attachment site. Analysis of synthetic amino acid sequences established a basic three-part signal for GPI anchoring: a cleavage/attachment domain that requires small amino acids at the first (GPI anchor attachment) and third positions but with little specificity at the middle position, a spacer domain of approximately 8-12 amino acids, and a hydrophobic domain of at least 11 amino acids. The ability to design a totally synthetic GPI anchoring signal will allow precise probing of the fine structure of this signal.
Turner AJ Essays Biochem 28: 113-27 (1994)Some membrane proteins are associated with the plasma membrane solely through a glycolipid moiety (GPI anchor). The GPI anchor is composed of a core structure of phosphatidylinositol attached to a glycan chain which, in turn, is attached to the C-terminus of the protein. The GPI-anchored protein can be released from the cell surface by the action of GPI-specific phospholipases C and D. In protozoa, GPI anchors represent the predominant mechanism for integrating cell-surface proteins into the lipid bilayer. Addition of a glycolipid anchor to a nascent protein requires a C-terminal hydrophobic signal sequence on the protein which is rapidly exchanged for a pre-assembled anchor. GPI anchors may have roles in protein targeting, cell signalling and in the uptake of small molecules (potocytosis). The human disease 'paroxysmal nocturnal haemoglobinuria' represents a defect in biosynthesis of the GPI anchor. Other lipid post-translational modifications of proteins are also recognized as important in regulating protein function (myristoylation, palmitoylation, prenylation).
Brewis IA; Ferguson MA; Mehlert A; Turner AJ; HooperNM J Biol Chem 270: 22946-56 (1995)The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 220.127.116.11) 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: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 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 alpha (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 Ser368. 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.
CD59 is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. Activation of the plasma complement (C) system leads to formation of a cytolytic membrane attack complex (MAC) composed of the terminal C components C5b, C6, C7, C8 and multiple copies of C9. While the main purpose of MAC is to destroy invading micro-organisms, the cells of the host have to be protected against self-destruction. Damaging effects of MAC are controlled by regulatory molecules which act either in the fluid phase or on the cell membrane.
The CD59 antigen (MACIF, MIRL, HRF-20 or protectin) is a major inhibitor of the MAC present on human cell membranes. CD59 inhibits complement lysis by preventing C5bă8-catalysed insertion and polymerization of C9 into cell membranes. The homologous restriction factor (HRF, C8bp, MIP) is another proposed inhibitor of MAC that binds to C5bă8. CD59 and HRF are anchored to cell membranes via a glycophosphoinositol (GPI) anchor. Soluble forms of HRF have been found in human urine, plasma and cerebrospinal fluid, where they appear to exert a limited complement inhibitory function..
The overall folding of CD59 resembles that of snake venom neurotoxins. The disulphide bonding pattern of the ten cysteines of CD59 defines a distinct domain that is found, for example, in the Ly-6 family of putative T-cell activation antigens and in triplicate in the urokinase-type plasminogen activator receptor. The CD59 domain has two antiparallel -sheets, one with three strands and another with two, that create a disc-shaped structure with extending loops.
The GPI anchor of CD59 is attached to the C-terminal Asn-77 residue and links the CD59 polypeptide to phospholipid. Initial biochemical analysis has indicated that the GPI-anchor structure resembles that of Thy-1 in having a core structure of ethanolamine-PO4-(▒Man1-2)Man1-2Man1-6Man(PO4-ethanolamine; ▒GalNAc)-GlcNH21-6myo-inositol.
The results reveal a whole spectrum of structures of both the oligosaccharide linked to Asn-18 and of the GPI anchor (at Asn-77). The latter include previously undescribed variants containing sialic acid. The soluble isoforms of CD59 retain their specific binding activity towards the TCCs but, because of the absent phospholipid tail, they only have a limited ability to inhibit MAC assembly on cell membranes.