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
alpha.
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

Replacement of the GPI anchor of Drosophila acetylcholinesterase with a transmembrane domain results in behavioral defects.

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.

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase:

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.

Glycosylphosphatidylinositol anchored recognition molecules that function in axonal fasciculation, growth and guidance in the nervous system.

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.

Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid.

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.

A mutant prion protein displays an aberrant membrane association when expressed in cultured cells.

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.

The association between GPI-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

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

A defect in GPI transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

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.

Construction of synthetic signals for GPIl anchor attachment. Analysis of amino acid sequence requirements for anchoring.

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.

PIG-tailed membrane proteins.

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).

Structures of the GPI anchors of porcine and human renal membrane dipeptidase

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 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: 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.

Structural composition and functional characterization of soluble CD59

Biochem. J. (1996) 316
Seppo MERI*, Timo LEHTO, Chris W. SUTTON, Jaana TYYNEL? and Marc BAUMANN

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 C5b?-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 C5b?. 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-(盡an1-2)Man1-2Man1-6Man(PO4-ethanolamine; 盙alNAc)-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.