Consensus Analysis of Signal Peptide Peptidase and Homologous Human Aspartic Proteases Reveals Opposite Topology of Catalytic Domains Compared with Presenilins

Abstract

The human genome encodes seven intramembrane-cleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP. The human genome encodes seven intramembrane-cleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP. Intramembrane-cleaving proteases cut proteins in transmembrane regions. They liberate fragments from dormant, membrane-bound precursor proteins, which typically exert downstream functions such as cell signaling and regulation (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar, 2Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (850) Google Scholar), immune surveillance (3Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar), and intercellular communication (4An F.Y. Sulavik M.C. Clewell D.B. J. Bacteriol. 1999; 181: 5915-5921Crossref PubMed Google Scholar, 5Lee J.R. Urban S. Garvey C.F. Freeman M. Cell. 2001; 107: 161-171Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). They are also thought to contribute to the development and propagation of pathological conditions such as Alzheimer's disease (6Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar) and hepatitis C virus infection (7McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar). Three families of proteases that promote intramembrane cleavage are known at present. These are a group of metalloproteases represented by the human site-2 protease (S2P) 1The abbreviations used are: S2P, site-2 protease; ER, endoplasmic reticulum; PBS, phosphate-buffered saline, PNGase F, N-glycosidase F; PS, presenilin; SPP, signal peptide peptidase; SPPL, SPP-like protease; TFPP, type IV prepilin peptidase; HA, hemagglutinin; Bicine, N,N-bis(2-hydroxyethyl)glycine. (8Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), a group of serine proteases represented by Drosophila melanogaster rhomboid-1 (9Urban S. Lee J.R. Freeman M. Cell. 2001; 107: 173-182Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), and a group of aspartic proteases with the human presenilins (PSs; PS1 and PS2) (10Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar) and signal peptide peptidase (SPP) (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) as prototypic members. PSs and SPP belong to the family of GXGD aspartic proteases (12Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Martoglio B. Golde T.E. Hum. Mol. Genet. 2003; 12: R201-R206Crossref PubMed Scopus (93) Google Scholar). Although the sequence homology is limited, sequences of PSs and SPP can be aligned almost throughout the entire length (14Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar). They are membrane proteins with multiple predicted transmembrane regions, two of which contain the active site motifs YD and GXGD, a unique trait specific for this protease family. These structural similarities make a case for a common catalytic mechanism. Indeed, a number of protease inhibitors, including aspartic protease transition state analogues, targeted PSs and SPP and inhibited both activities (15Weihofen A. Lemberg M.K. Friedmann E. Rueeger H. Schmitz A. Paganetti P. Rovelli G. Martoglio B. J. Biol. Chem. 2003; 278: 16528-16533Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Despite common features, there are major differences between PSs and SPP. PSs undergo endoproteolysis for activation and appear to act as proteolytic subunits of a multiprotein complex called γ-secretase (16Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar, 17De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 18Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). For SPP activity, in contrast, there is no indication for a requirement neither for endoproteolytic activation nor for additional components (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). One of the most striking differences, however, is the apparent opposite orientation of the catalytic domains within the plane of the membrane (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Transmembrane regions containing the catalytic aspartates have opposite orientation in PSs when compared with corresponding transmembrane regions in SPP. Because PSs are known to catalyze intramembrane cleavage of several type I anchored membrane proteins (e.g. β-amyloid precursor protein and Notch-1) (19Kopan R. Ilagan M.X.G. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (499) Google Scholar), whereas SPP cuts type II-oriented transmembrane peptides (e.g. signal sequences, hepatitis C virus core protein precursor), the orientation of the respective catalytic site seems predictive for the orientation of the relevant substrates. Besides the two PSs and SPP, the human genome contains four additional candidate GXGD proteases, which have been recognized by data base searching (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar, 14Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar, 20Grigorenko A.P. Moliaka Y.K. Korovaitseva G.I. Rogaev E.I. Biochemistry (Moscow). 2002; 67: 826-835Crossref PubMed Scopus (56) Google Scholar). These proteins, referred to as PSH (for PS homologues) (14Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar), IMPAS (for intramembrane proteases) (20Grigorenko A.P. Moliaka Y.K. Korovaitseva G.I. Rogaev E.I. Biochemistry (Moscow). 2002; 67: 826-835Crossref PubMed Scopus (56) Google Scholar), or SPPLs (for SPP-like proteases) (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar), have no identified function. Like the PSs and SPP, they are proteins with multiple predicted transmembrane regions. SPP and SPPLs share high homology particularly in the C-terminal half of the molecules, which contain the catalytic motifs and the conserved sequence QPALLY (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar, 14Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar). Because of this structural homology, it is likely that these proteins have the same enzymatic activity, namely that of an intramembrane-cleaving aspartic protease. The divergence observed in the N-terminal portions may be a determinant of their individual function. In the present study we have investigated the topologies of SPP and the homologous human SPPLs. After cloning the respective cDNAs, we expressed the proteins in a cell-free in vitro system and in living cells, and we determined the topological function of the most N-terminal hydrophobic region and the localization of the N- and C-terminal portions. Furthermore, we determined the localization of the hydrophilic loops that connect the two transmembrane regions containing the catalytic aspartate residues. The topology of the latter is crucial for the future search for substrates of the SPPLs, because experimental data on PSs and SPP suggest that the topology of the catalytic domain correlates with the membrane orientation of the respective substrates. Cloning SPPLs and Plasmid Constructs—Total RNA was isolated from SH-SY5Y and HEK293 cells using and with the of sequence (for and SPPL3 using and and and was from a human and from a using the and and and The and the and by by RNA and from and the of The of all was determined with an analysis was as Y. J.R. T. A.P. A. A. S. A. 2002; PubMed Scopus Google Scholar). In of RNA was used to that was used as a to was and to and the human from the of the to the in the of After and with an was used to are the from by two and two In and for the respective region was with using as and a with to a at the M.K. Martoglio B. 2003; PubMed Scopus Google Scholar). for a containing the and sequence by for a site for and the was in vitro with RNA at in the of RNA structure in of containing and and, of from and to B. S. B. Cell Scholar). for at with and to M.K. Martoglio B. 2003; PubMed Scopus Google Scholar). in and cells at in in containing and the using In of and of the used for and cells for with containing the cells with and in containing and protease For with N-glycosidase F, at for to the and cells on in the cells with PBS, for with at and with by for at in and cells with and with in at for cells the same was to the For with and with the and or the of in for at After with PBS, the cells for at with and in After with PBS, with and with using a and by using C for and SPP and C for SPPL2a, and a C for M.K. Martoglio B. 2003; PubMed Scopus Google Scholar). proteins by a For proteins by and to which with in with and was by of N-terminal extensions investigated in of used for the in vitro For the N-terminal the hydrophilic N-terminal the hydrophobic region the downstream by cell-free in vitro In SPP and SPPL3, the downstream region also contains transmembrane regions and transmembrane for to an SPPL3 containing an additional site for at the of in a cell-free in vitro system in the of for protein translocation and to the respective proteins modified with or from which a signal sequence been of catalytic of of SPP and SPPLs, which contain an additional site for within the catalytic transmembrane containing the catalytic of as from in and of and of SPP and SPPLs in with PNGase for containing the additional site in the catalytic the type and of SPP and the presenilins PS1 and and the type IV prepilin are of the GXGD family of aspartic proteases (12Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Martoglio B. Golde T.E. Hum. Mol. Genet. 2003; 12: R201-R206Crossref PubMed Scopus (93) Google Scholar). the data a number of additional candidate GXGD proteases present in the genome of of all are proteins with function. analysis these sequences SPP and its human homologues appear to be of containing SPP, containing SPPL3, and a family containing SPPL2a, -2b, and These proteins to the PSs and that SPP and its homologues may have structural and substrate that from the In to the of SPP and SPPLs, we the from human cell and the sequences in the data base using we determined the of SPP and in human The the of for SPP, SPPL2a, -2c, and SPPL3 in all major human a of the respective proteins For the was that is We investigated the sequences of the human proteins SPP, SPPL2a, -2b, and -2c, and SPPL3 by the sequence analysis and The of membrane protein topology J. B. G. 2000; PubMed Scopus (93) Google Scholar) of SPP and SPPLs predicted a common of hydrophobic predicted transmembrane regions two of which contain the conserved catalytic YD and GXGD I and Furthermore, the sequences of SPPL2a, -2b, -2c, and contain to their an additional hydrophobic region for which the function of an endoplasmic signal B. B. Trends Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In all proteins, an hydrophilic region containing for the signal sequence In contrast, neither an N-terminal signal sequence nor an hydrophilic in the N-terminal of the protein is predicted for SPP and SPPL3 I and of predicted transmembrane regions in a of SPP and SPPLs in the topology of SPP and SPPLs, we of proteins in We cells with proteins the of the the SPPL2a, -2b, and -2c, we expressed proteins containing a C-terminal and from the SPP we expressed a containing an of the C-terminal signal J. Cell Biol. Google Scholar). of proteins was by Because all investigated proteins contain multiple for we also with N-glycosidase toward PNGase is of the of and of the respective region toward the exoplasm. of SPP in two of corresponding to SPP and of The latter most likely the of SPP to a by Nyborg Jansen K. M.S. Golde T.E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), on in to with PNGase F, which the apparent of the to and is with the of two from the N-terminal for SPPL2a, -2b, and For these proteins we of which all to with PNGase (for the analysis of also the system in The observed in are in with the of the number of from the N-terminal to for SPPL2a, for and or for most N-terminal is to the is typically of SPPL3 a as by the of toward PNGase The high to the protein as by with in vitro SPPL3 for SPPL3 a at was particularly when cells conditions no Taken together, these that the N-terminal hydrophilic domains of SPP and SPPLs are exposed toward the and, with the of the of SPPL3, modified with N-glycans. of N-terminal the localization of the termini and the topology of the hydrophobic regions in by using a cell-free in vitro The most N-terminal hydrophobic region of a membrane protein to the membrane and the a hydrophobic region may function as signal sequence that is from the of the the membrane been B. B. Trends Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the protein located the cell in the exoplasm. the hydrophobic region may function as a signal anchor sequence that also and membrane is and functions as a membrane anchor sequences can the in a type I the N-terminal is the and exposed toward the whereas the C-terminal in the or in a type the in the whereas the C-terminal B. B. Trends Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). For SPP, we have that its hydrophobic region functions as a type I signal anchor sequence (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). of the N-terminal hydrophilic of the SPP is by the of at the two for present in this region and the N-terminal regions of the SPPLs are as the of SPP, we of the cell-free in vitro system for protein and for membrane G. B. J. Cell Biol. 67: PubMed Scopus Google Scholar). In the of we of for the N-terminal hydrophilic the hydrophobic the downstream analysis the function of a signal sequence for the hydrophobic region of SPPL2a, -2b, and -2c, and for within the downstream residues. Indeed, when the respective in the in vitro of in the of when compared with These when the J.T. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was to the The major in these latter however, than the in the of a signal sequence These data the namely that SPPL2a, -2b, and -2c contain an N-terminal signal sequence, and demonstrate that N-terminal domains of are and therefore facing the exoplasm. of the sequence of SPPL3 the function of a signal anchor sequence for the hydrophobic like for SPP, and for within the downstream in the N-terminal hydrophilic the SPPL3 was in the of the as the in the of in the of the the apparent was that the hydrophobic region of SPPL3 functions as a type I signal anchor sequence, and the downstream hydrophilic and be facing the the topology of this transmembrane we a site for the hydrophilic N-terminal in the of was translocation the of the of we to the localization of the the hydrophilic which the two transmembrane regions containing the catalytic aspartate hydrophobic regions and In SPP, this is predicted to be exposed toward the whereas in the presenilins the respective regions are located in the A. Thinakaran G. T. M. Selkoe D.J. M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, I. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, I. S. A. PubMed Scopus Google Scholar, T. A. M. K. K. Y. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the catalytic loops of SPP and SPPLs are exposed toward the or the we a site for the hydrophilic region and we expressed proteins in Because can to than from transmembrane regions G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. J. Biol. 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SPP contains a which can function when exposed toward the cytosol. however, of the membrane for the C-terminal of the SPP, whereas the protein was The protein was by the membrane and of all with the C of SPP as exposed toward the like the of the protein I. D. D. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), from which we the same for the SPPLs. In all the C-terminal of the membrane and be by whereas the protein was by the membrane and with These that the C-terminal of SPP and SPPLs are all exposed in the cytosol. Taken with the we that SPP and SPPLs are candidate aspartic proteases the same principal structure and, like SPP, are to promote intramembrane of transmembrane regions, which the membrane in type topology is a for experimental on the of membrane Here we have the structural of the intramembrane-cleaving protease SPP and its four human homologues SPPL2a, -2b, -2c, and -3. with a of membrane protein we have determined in vitro and in living cells the of the termini and topological of most N-terminal hydrophobic regions, the orientation of catalytic and the of the C We that SPP and SPPLs share the same principal topology and appear to be to cleave a type II-oriented transmembrane region of substrate is in to the presenilins, catalytic domains have the opposite orientation within the membrane and, are to cleave type transmembrane regions. the for the search of candidate substrates and future of functions of these membrane proteases. a membrane protein is thought to its topology by the transmembrane region and of in opposite orientation S. 1999; PubMed Scopus Google Scholar, J. 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G. 2000; PubMed Scopus (93) Google Scholar). topology of membrane proteins however, by the that of these proteins have N-terminal signal which can the of in topology of the most N-terminal transmembrane region for to an predicted orientation for the transmembrane regions. a hydrophobic core of a domain may be as a transmembrane and hydrophobic containing or a region can be it is crucial to the predicted topology by experimental in the of topology as compared with data by apparent in the present The and for the signal sequence of the localization for the N-terminal domain was For predicted type I orientation for the hydrophobic region determined signal and as a the orientation of the transmembrane regions was predicted a type signal anchor sequence at the termini of SPP and SPPL3, whereas our a type I signal anchor a the topology for the downstream catalytic loops and the C termini opposite the determined the the topology of SPP and SPPLs was when the with our experimental the topology of the most N-terminal transmembrane regions and the of the C termini on the topology of the catalytic loops with the Taken together, our a structural SPP and its candidate proteases, SPPL2a, -2b, -2c, and have transmembrane regions with the hydrophilic N-terminal domain and catalytic exposed toward the whereas the C is located in the Intramembrane-cleaving proteases contain multiple hydrophobic regions that are to a proteolytic domain within the plane of the We that this type of proteases, like all membrane proteins, have a orientation in the membrane and are likely to cleave transmembrane regions of of type I or type orientation this can be in the intramembrane-cleaving metalloproteases N.G. R.B. Brown M.S. Goldstein J.L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) with its J. Bacteriol. 2000; PubMed Scopus Google Scholar), and the aspartic proteases SPP (11Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) with the PS1 A. Thinakaran G. T. M. Selkoe D.J. M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, I. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, I. S. A. PubMed Scopus Google Scholar, T. A. M. K. K. Y. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The hydrophobic regions containing the respective catalytic are predicted to have opposite transmembrane orientation in and SPP, when compared with of and In with the opposite orientation of catalytic the transmembrane regions of known respective substrates have opposite orientation by and SPP have type whereas by and PS1 have type I Because the topology of the catalytic domain of an intramembrane-cleaving protease seems to be the in the transmembrane orientation of its our that -2b, -2c, and like SPP, cleave substrate proteins within a type II-oriented transmembrane In our structural of SPP that are common to its four human homologues, SPPL2a, -2b, -2c, and -3. their substrate we can search for candidate substrates of the SPPLs. These can be type membrane proteins or type II-oriented transmembrane regions of membrane proteins M.K. 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