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

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell- free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.     

Cell-free processing of nascent proteins destined to be linked to the plasma membrane by a phosphatidylinositol-glycan anchor.

Certain proteins are anchored to the outer plasma membrane by a phosphatidylinositol-glycan (PI-G) linker. Nascent forms of PI-G anchored proteins contain both NH2- and COOH-terminal signal peptides. The function and structural requirements of the COOH-terminal signal peptide as discussed and some studies on the cell-free processing of a nascent protein to its mature PI-G tailed form are presented.     

Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site.

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophosphatidylinositol (GPI) membrane anchor in a process involving proteolytic removal of 17-31 COOH-terminal residues. Previous work suggested that two elements are required for anchor addition, a COOH-terminal hydrophobic domain (the GPI signal) and an element located NH2-terminal to it, postulated to be the cleavage/attachment site. Using [3H]ethanolamine (a component of the anchor) to tag the COOH terminus, we isolated and sequenced a COOH-terminal tryptic peptide, thereby identifying Ser-319 as the COOH-terminal residue attached to the GPI anchor. This indicates that a 28-residue peptide is removed during processing and localizes the cleavage/attachment site precisely to the region previously shown to be required for anchor attachment (between 10 and 20 residues NH2-terminal to the hydrophobic domain). Since DAF contains multiple cryptic cleavage/attachment sites, we used a GPI-linked human growth hormone-DAF fusion to study the structural requirements for cleavage/attachment. Our results show that while sequences immediately NH2-terminal to the attachment site are not required for anchor addition, deletion of Ser-319 abolishes both anchor attachment and transport to the cell surface. Systematic replacement of the attachment site serine with all possible amino acids indicated that alanine, aspartate, asparagine, glycine, or serine efficiently support GPI anchor attachment while valine and glutamate are partially effective. All other substitutions including cysteine (permitted at the attachment site in other GPI-anchored proteins) abolish both GPI anchor attachment and transport to the cell surface, resulting in accumulation of uncleaved fusion protein in internal compartments (endoplasmic reticulum and Golgi). These results support the general rule that the residue at the cleavage/attachment site must be small. Further, addition of a GPI anchor appears to be necessary for transport to the cell surface in transfected COS cells.     

Parallel effects of signal peptide hydrophobic core modifications on co-translational translocation and post-translational cleavage by purified signal peptidase

The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage.     

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.     

Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide

Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

US2, a human cytomegalovirus-encoded type I membrane protein, contains a non-cleavable amino-terminal signal peptide.

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.     

Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence.

Surfactant protein D (SP-D) is a carbohydrate-binding glycoprotein containing a collagen-like domain that is synthesized by alveolar type II epithelial cells. The complete primary structure of rat SP-D has been determined by sequencing of a cloned cDNA. The protein consists of three regions: an NH2-terminal segment of 25 amino acids, a collagen-like domain consisting of 59 Gly-X-Y repeats, and a COOH-terminal carbohydrate recognition domain of 153 amino acids. There are 6 cysteine residues present in rat SP-D: 2 in the NH2-terminal noncollagenous segment and 4 in the COOH-terminal carbohydrate-binding domain. The collagenous domain contains one possible N-glycosylation site. The protein is preceded by a cleaved, NH2-terminal signal peptide. SP-D shares considerable homology with the C-type mammalian lectins. Hybridization analysis demonstrates that rat SP-D is encoded by a 1.3-kilobase mRNA which is abundant in lung and highly enriched in alveolar type II cells. Extensive homology exists between rat SP-D and bovine conglutinin.     

Biosynthesis of human preproapolipoprotein A-II

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.     

Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides.

The Bacillus subtilis alpha-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis alpha-amylase signal peptides of different lengths and the mature thermostable alpha-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable alpha-amylases and by in vivo pulse-chase experiments. In B. subtilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and alpha-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin

Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and this proteolytic site is shared by a majority of other L1 family members. We provide evidence with furin-specific inhibitor experiments, by site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin-deficient LoVo cells that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain-specific antibodies suggest that the resulting NH(2)- and COOH-terminal cleavage fragments interact with each other and that this interaction provides a means for the NH(2)-terminal fragment to be tethered to the membrane. Furthermore, in S2 cell aggregation assays we show that the NH(2)-terminal fragment is necessary for homophilic adhesion and that cells expressing only the transmembrane COOH-terminal fragment are non-adhesive. However, tethering of exogeneously provided Tractin NH(2)-terminal fragment to S2 cells expressing only the COOH-terminal fragment can functionally restore the adhesive properties of Tractin.