C-terminal Signal Peptide Research Articles

Proteasome and thiol involvement in quality control of glycosylphosphatidylinositol anchor addition.

Improperly processed secretory proteins are degraded by a hydrolytic system that is associated with the endoplasmic reticulum (ER) and appears to involve re-export of lumenal proteins into the cytoplasm for ultimate degradation by the proteasome. The chimaeric protein hGHDAF28, which contains a crippled glycosylphosphatidylinositol (GPI) C-terminal signal peptide, is degraded by a pathway highly similar to that for other ER-retained proteins and is characterized by formation of disulphide-linked aggregates, failure to reach the Golgi complex and intracellular degradation with a half life of approximately 2 h. Here we show that N-acetyl-leucinal-leucinal-norleucinal, MG-132 and lactacystin, all inhibitors of the proteasome, protect hGHDAF28; hGHDAF28 is still proteolytically cleaved in the presence of lactacystin or MG-132, by the removal of approximately 2 kDa, but the truncated fragment is not processed further. We demonstrate that the ubiquitination system accelerates ER-degradation of hGHDAF28, but is not essential to the process. Overall, these findings indicate that GPI quality control is mediated by the cytoplasmic proteasome. We also show that the presence of a cysteine residue in the GPI signal of hGHDAF28 is required for retention and degradation, as mutation of this residue to serine results in secretion of the fusion protein, implicating thiol-mediated retention as a mechanism for quality control of some GPI signals. Removal of the cysteine also prevents inclusion of hGHDAF28 in disulphide-linked aggregates, indicating that aggregate formation is an additional retention mechanism for this class of protein. Therefore our data suggest that an unpaired terminal cysteine is the retention motif of the hGHDAF28 GPI-processing signal and that additional information may be required for efficient engagement of ER quality control systems by the majority of GPI signals which lack cysteine residues.

Mutational analysis of the C-terminal signal peptide of bovine liver 5′-nucleotidase for GPI anchoring: a study on the significance of the hydrophilic spacer region

Bovine liver 5′-nucleotidase is a GPI-anchored protein whose Ser 523 attaches to GPI as the ω-site. For GPI-modification, pro-protein of the enzyme possesses a signal peptide at the C-terminus, comprising a hydrophilic spacer sequence of 8 amino acid residues and the following hydrophobic region of 17 amino acid residues. The C-terminal signal peptide is replaced by GPI on a luminal leaflet of endoplasmic reticulum. To characterize the C-terminal signal peptide for GPI modification, we constructed a series of deletion and elongation mutant genes, altering length of the hydrophilic spacer sequence by site-directed mutagenesis. Systematic deletion and Ala insertion of the sequence showed that the sequence of 6–14 residues were compatible for GPI modification. For GPI transfer to the pro-protein, the optimum length of spacer sequence would be 8, being consistent with natural selection. The spacer sequence may play a role for leading the ω-residue correctly to the active site of putative GPI transamidase. The elongation of the spacer is more permissible than deletion. Nevertheless, the length of the spacer sequence may influence efficiency of GPI modification by its positive or negative control.

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

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 N-terminal signal sequence and a C-terminal signal peptide. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-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 of up 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 pQ22) and provided the processed C-terminus as M1004 for the native, GPI-unmodified form of axonin-1.

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (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. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.

The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants.

The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCD14). Previous studies indicate that monocytes produce sCD14 by limited proteolysis of the membrane-bound receptor. In this study we demonstrate that human monocytes also produce sCD14 by a protease-independent mechanism. To investigate the molecular nature of this second pathway we studied sCD14 formation in the monocytic cell line Mono Mac 6 (MM6) and in CD14 transfectants. Both MM6 and the CD14 transfectants constitutively produce sCD14 by a protease-independent mechanism. Structural analysis of sCD14 produced by the CD14 transfectants reconfirmed the presence of the COOH terminus predicted from the cDNA. Since glycosylphosphatidylinositol anchor attachment is associated with the removal of a hydrophobic C-terminal signal peptide, our finding demonstrates that the transfectants secrete sCD14 which escaped this posttranslational modification. Identical results obtained for sCD14 derived from peritoneal dialysis fluid of a patient with kidney dysfunction show the in vivo relevance of this pathway for sCD14 production.

Biosynthesis and processing of the glycosylphosphatidylinositol anchor in mammalian cells

Many T cell surface proteins are attached to the cell membrane by glycosylphosphatidylinositol (GPI) anchors, which may be involved in cell signaling, protein targeting or protein release. Proteins destined to be GPI-anchored have both N- and C-terminal signal peptides, which permit the nascent polypeptides to transverse the endoplasmic reticulum and to be transferred to preformed GPI anchors in a transamidase reaction. The biosynthetic pathway of the GPI anchor in mammalian cells has been elucidated using a panel of T cell mutants that cannot express GPI-anchored proteins on their cell surfaces. The first step in anchor biosynthesis is the transfer of N-acetylglucosamine (GlcNAc to a phosphoinositol (PI) acceptor to form GlcNAc-PI, which is then deacetylated to form GlcN-PI. Subsequently, a fatty acid is added to the inositol ring and three mannose residues are transferred to the elongating GPI core. Finally, ethanolamine phosphates are transferred to the mannose residues. Three human cDNAs encoding for GPI synthases (Classes A, F and H) have been identified using expression cloning technique. Mutation in the X-linked class A gene was shown to be the cause of paroxysmal nocturnal hemoglobinuria, an acquired disease affecting hematopoietic stem cells in which the abnormal cells are deficient in GlcNAc-PI formation. The potential involvement of GPI-anchored proteins or GPIs in T cell activation is also discussed.