Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

Kodukula, Krishna; Gerber, Louise; Amthauer, Rodolfo; Brink, Larry; Udenfriend, Sidney

doi:10.1083/jcb.120.3.657

Cited by 121 publications

(90 citation statements)

References 32 publications

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“…We introduced additional prolines, in view of suppressing glypiation. Our results are perfectly consistent with the identification of the natural site as Gly 14 , but we found that it was not possible to suppress completely helix and the site Chimeric constructs are shown at the top. The GPI addition signal is composed of intervening regions (located between the cysteine and the hydrophobic region) and hydrophobic regions from rat (r) or Torpedo (t).…”

Section: Resultssupporting

confidence: 81%

“…The structural requirements of the C-terminal signal peptides that induce GPI addition have been investigated extensively by the groups of Caras and Udenfriend, by mutagenesis of the "decay-accelerating factor" (9 -12) and of placental alkaline phosphatase (13)(14)(15)(16)(17). They showed that glypiation requires a C-terminal hydrophobic sequence and an upstream cleavage/addition site (2,13).…”

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confidence: 99%

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Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Coussen

Ayon

Goff

et al. 2001

Journal of Biological Chemistry

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We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet / ؉ 1/ ؉ 2, particularly ؊ 1. 5) Glypiation was not affected by the closeness of the site to the ␣ 10 helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of . 7) Glypiation was not affected by the presence of an N-glycosylation site at or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region.

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Section: Resultssupporting

confidence: 81%

mentioning

confidence: 99%

Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Coussen

Ayon

Goff

et al. 2001

Journal of Biological Chemistry

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“…Studies in mammalian cells [5,6] and in Saccharomyces cereisiae [7] have used variants of C-terminal sequences to define the elements that are necessary and sufficient for GPI attachment (reviewed in [8]). In order from the C-terminus, these consist of a stretch of 8-20 hydrophobic residues, a short region of amino acids rich in charged residues and proline, and a domain of three small amino acids, the most N-terminal of which is the attachment or ω site [9].…”

Section: Introductionmentioning

confidence: 99%

Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

Doering

Schekman

1997

Biochemical Journal

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The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cere isiae protein Gas1p. Maturation of fusion proteins expressed in i o required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in itro for use in cell-free studies of glycolipid

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“…Several previous studies have postulated empirical rules to define GPI-attachment signal sequences from a cDNA-sequence. 21 With regard to such a possible signal, peptide Met-983 has not been suggested as a GPIattachment site.…”

Section: Identification Of N-and C-terminal Sequences and Heterogeneitymentioning

confidence: 99%

Mass spectrometric characterisation of primary structure, sequence heterogeneity, and intramolecular disulfide loops of the cell adhesion protein axonin-1 from chicken

Denzinger

Przybylski

Savoca

et al. 1997

Eur. J. Mass Spectrom.

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Axonin-1 is an axon-associated cell adhesion protein which exists as a membrane-attached form via a glycosylphosphatidyl-inositol anchor (GPI) and a secreted form. Axonin-1 promotes and regulates neurite outgrowth and plays a critical role as a growth cone sensor molecule in axonal pathways. The cDNA of axonin-1 from chicken codes for 1036 amino acids, including a hydrophobic Nterminal signal sequence and a C-terminal signal peptide. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) of native chicken axonin-1 showed an apparent molecular weight of 130-140 kDa. The characterisation of intact axonin-1 by matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) yielded molecular masses of 122,750 and 125,100 Da which are consistent with the GPI-modified and an unmodified secreted form. The mass difference between the calculated molecular mass based on the amino acid sequence (107,640 Da) suggests modifications ofup to 15 kDa which are assumed to be mainly due to glycosylation. In this study, the detailed structural characterisation of axonin-1 in its secreted GPI-anchorless form was carried out. Mass spectrometric peptide mapping analyses using the endoproteinases Lys-C and Asp-N revealed the complete amino acid sequence, the N-and C-terminal heterogeneities and, in conjunction with reductive cleavage, the intramolecular disulphide-loop pattern. Furthermore, the preliminary results on the location of N-glycosylation sites could be obtained. In addition, proteolytic fragments were isolated by high performance liquid chromatography (HPLC), identified by MALDI-MS and further digested with trypsin. These results revealed the presence of a blocked N-terminus (pyroglutamyl pQ 22 ) and provided the processed C-terminus as M 1004 for the native, GPI-unmodified form of axonin-1.

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Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

Cited by 121 publications

References 32 publications

Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system

Mass spectrometric characterisation of primary structure, sequence heterogeneity, and intramolecular disulfide loops of the cell adhesion protein axonin-1 from chicken

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