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The amoebapore superfamily
Biochimica et Biophysica Acta 1469 (2000) 87^99 www.elsevier.com/locate/bba
The amoebapore superfamily Yufeng Zhai, Milton H. Saier Jr. * Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA Received 28 April 2000; accepted 8 June 2000
Abstract Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Amoebapore; Saposin; NK-lysin; Countin; Protozoan; Membrane; Pore; Disease; Molecular phylogeny
1. Introduction Amoebiasis is a disease caused by the protozoan parasite Entamoeba histolytica. The most prominent pathogenic feature of this disease is the powerful ability of the protozoan to kill target cells, resulting in massive tissue destruction [1]. Three amoebapore isoforms termed amoebapores A, B and C have been isolated. They form ion channels in membranes by oligomerization, and thus a¡ect membrane function and eventually result in lysis of the target cell [2]. Based on sequence and structural similarities, amoebapores are homologous to many other proteins, including saposins (SAPS) A, B, C and D [3], T-cell cytolytic proteins such as NK-lysins [4] and granuly-
* Corresponding author. Fax: 1-858-534-7108; E-mail: [email protected]
sins [5], pulmonary surfactant-associated proteins [6], acid sphingomyelinases (ASMs) [7,8], acyloxyacyl hydrolases (AOAH) [9], and swaposins which exhibit the SAP motif in plant aspartic proteases (AP) but with swapped N-terminal and C-terminal halves of the motif [10]. All of the proteins mentioned above and their homologous domains are termed saposinlike proteins, since SAPs A^D were the ¢rst proteins of this family to be characterized. Most of the proteins which comprise the amoebapore superfamily contain six conserved cysteine residues which form three disul¢de bridges. The NKlysins which contain ¢ve cysteines that form two disul¢de bridges represent the only exceptions to this structural pattern. These disul¢de bridges are in part responsible for the extraordinary thermostability of these proteins. For example, amoebapore A retains its activity after heating at 100³C for 15 min under non-reducing conditions [11].
0304-4157 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 5 7 ( 0 0 ) 0 0 0 0 3 - 4
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Interestingly, there is another protein, countin, which we here show to be a new member of the amoebapore superfamily. It is a newly characterized protein [12] which allows groups of slime mold cells to recognize their numbers and thereby regulate aggregate size. Like most other members of the amoebapore superfamily, countin contains a saposin-like domain with six conserved cysteine residues. In this paper we identify all currently sequenced members of the amoebapore superfamily, subdivide them into eight families (including the newly identi¢ed `countin' family), present the sequence alignments, and discuss the structures and functions of these proteins. We provide evidence that all these proteins including countin are evolutionarily related to each other. 2. The pore-forming amoebapore (PFA) family (Table 1 and Fig. 1) Amoebapores, pore forming peptides of the protozoan E. histolytica, are the cause of tissue destruction in the human host. Experimental data indicate that amoebapores create ion channels in membrane by oligomerization [2]. Three isoforms, termed amoebapore A, B and C, with mature proteins of 77-residues in length, exist in cytoplasmic granules of the amoeba. They display antibacterial activity and are instrumental in preventing the growth of phagocitized bacteria. Secondary structural predictions suggested that
amoebapore has four amphipathic alpha-helical domains [14] stabilized by six cysteines which form three disul¢de bridges (Fig. 1). In Fig. 1 and in subsequent ¢gures in this paper, the sequences of all or most members of a family are aligned; however, if two or more homologues show very high percent identities, only one of them is shown. The 3D structure of NK-lysin has been solved by NMR [15], and the structure of amoebapore has been postulated based on homology modeling methods. The theoretical structure, which has ¢ve K-helices arranged in two layers, provides insight into possible structure^function relationships [16]. The results show that amoebapores A, B and C exhibit hydrophobic accessible surface areas that correspond to about 26, 32 and 32% of the total surface, respectively. The largest hydrophobic areas overlap helices 1, 3 and 5, creating hydrophobic grooves between the two layers. The hydrophobic cores between the two layers are loosely packed according to cavity analysis. Besides these features, there is no biased charge distribution in any of the isoforms. This feature differs from that suggested for NK-lysin, implying a di¡erent mechanism of membrane interaction. The hypothesized mechanism suggests a conformational rearrangement and concomitant membrane insertion of helices 2 and 3 after initial lipid binding and oligomerization. The resultant transmembrane pore comprises a permeability barrier and may eventually lyse the target cell. All of the nearest amoebapore subfamily homo-
Table 1 Proteins of the PFA family Source organism
Accession number
GI number
Database description
PFPB Ehi
Length 96
E. histolytica
Q24824
2498767
PFPB Edi
96
E. dispar
AAF04195
6119727
PFPA Ehi
98
E. histolytica
P34095
464366
PFPN Ehi
97
E. histolytica
Q07831
730303
PFPC1 Ehi PFPC2 Ehi
88 101
E. histolytica E. histolytica
BAA22028 Q24825
2351011 2498768
PFPC Edi
101
E. dispar
AAF04196
6119729
pore-forming peptide amoebapore B precursor pore-forming protein isoform B precursor pore-forming peptide amoebapore A precursor nonpathogenic pore-forming peptide precursor amoebapore C pore-forming peptide amoebapore C precursor pore-forming protein isoform C precursor
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Fig. 1. Sequence alignment of pore-forming amoebapore family proteins. In this and the subsequent ¢gures, protein abbreviations are as indicated in Table 1 or in the corresponding table. Shaded residues are the conserved cysteines. An asterisk below the alignment indicates a fully conserved residue, while one or two dots indicate distant and close similarities, respectively.
logues retrieved from the database using the BLAST [17] search tool are presented in Table 1. These proteins are from Entamoeba histolytica and E. dispar. The two homologues obtained from E. dispar are closely related to those from E. histolytica. Both species live as commensals in the lower human intestine. However only the latter causes disease [18]. In vitro, both amoeba can kill host cells, but E. dispar is approximately threefold less e¡ective than E. histolytica [19]. 3. The saposin (SAP) family (Table 2 and Fig. 2) The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called SAPs which are generated from common precursors called prosaposins [20]. Selective SAP de¢ciencies are known only for Sap B and C. A de¢ciency in Sap B or C causes increased storage of sulfatides [21,22] and glucosylcermides [23]. De¢ciencies of Sap A or D have not been reported. Although their physiological functions have not been ¢rmly established, one hypothesis is that Sap A stimulates the hydrolysis of glucosyl- and galactosylceramides [24,25]. Sap D is likely to stimulate degradation of ceramides since its addition to the culture medium of ¢broblasts from patients with a prosaposin de¢ciency led to a decrease in accumulated ceramide [26]. Mature SAPs are small proteins of about 80 amino acids that share several structural properties which include the six conserved cysteines. Although their involvement in the degradation of sphingolipids is
well established, their mechanisms of action are still under debate [27]. It has been assumed that the mode of action of Sap B is based on its capacity to solubilize sulfatides to make them accessible to the degradative action of arylsulfatases. As to Sap C, the physiological activator of glucosylceramidase, it is thought that it enhances enzyme activity by forming an active complex with the enzyme [20]. Recently this hypothesis has been questioned. Sap C may instead promote glucosylceramidase localization to lipid surfaces [28]. Sap A also stimulates the glucosylceramidase activity probably by inducing a conformational change in the enzyme. However, detailed information regarding its mechanism of action is lacking. It was hypothesized that the action of Sap D depends on its interaction with the enzyme rather than with the ceramide substrate [29]. Table 2 and Fig. 2 present a list of the SAP proteins retrieved from the databases and the alignment of their sequences, respectively. 4. The NK-lysin/granulysin family (Table 3 and Fig. 3) NK-lysin and granulysin are anti-bacterial peptides produced by pig natural killer cells [30] and human cytolytic lymphocytes [5], respectively. NKlysin shows antibacterial activity as well as cytolytic activity toward certain cancer cells. Granulysin exhibits a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. Multiple sequence alignments show that
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Table 2 Proteins of the SAP family Length
Source
Organism
Accession number
GI number
Description
pig
Sus scrofa
P81405
3914938
slime mould cow
D. discoideum Bos taurus
BAA32237 P26779
3402901 134216
guinea pig
Cavia porcellus
P20097
134217
524
human
H. sapiens
P07602
134218
518 554 554 556 402
chicken rat house mouse mouse worm
Gallus gallus Rattus sp. M. musculus Mus sp. C. elegans
AAF05899 AAB36233 AAA92567 AAB31059 AAB37548
Sap B (cerebroside sulfate activator) Sap A Sap C (co-L-glucosidase) Sap C (co-L-glucosidase) proactivator polypeptide precursor prosaposin prosaposin sulfated glycoprotein prosaposin weak similarity to mouse sphingolipid activator protein
Sap Ssc
79
Sap Ddi Sap Bta
143 80
Sap Cpo
81
Sap Hsa Sap Sap Sap Sap Sap
Gga Rsp Mmu Msp Cel
these proteins have invariant cysteine residues as do the SAPs and amoebapores, but the ¢rst cysteine in granulysin is replaced by tyrosine as shown in Fig. 3 which allows it to form only two disul¢de bridges. This di¡erence is thought to be of little signi¢cance with respect to protein stability [16]. The 3D structure of NK-lysin, recently solved by NMR spectroscopy, provides a template for estimation of the structures of other SAP-like proteins such as granulysin and amoebapore [15]. There is a groove
6224674 1336844 881390 557967 1065921
at the interface of helices 1, 3 and 5, and the protein exhibits small hydrophobic patches near the C-terminal surface of helix 1. Hydrophobic residues occupy about 17% of the accessible surface of this protein. NK-lysin has a net charge of 6+, and most of these charges are arranged in an equatorial belt around the molecule in a vertical position of helix 1. This belt divides a more negatively charged half of the molecule from the other half around the N- and C-termini which contain few acidic residues. These character-
Fig. 2. Sequence alignment of SAP family proteins.
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Table 3 Proteins of the NK-lysin family TCAP Hsa Gran Hsa NKG5 Hsa NKL1 Ssc NKL2 Ssc
Length
Source
Organism
Accession number
GI number
Description
129 145 145 129 78
human human human pig pig
H. sapiens H. sapiens H. sapiens S. scrofa S. scrofa
A27562 NP_006424 CAA38035 Q29075 2392473
88664 5453784 35065 2498644 2392473
T-cell activation protein granulysin NKG5 product NK-lysin precursor NK-Lysin
istics of NK-lysin imply a possible mechanism of membrane interaction. The N-terminal and C terminal residues, localized to helices 1 and 5, respectively, dip into the membrane; the positively charged belt interacts with the phospholipid head groups, and the other half of the protein is exposed to the solvent. The potential for pore formation remains unclear, and electrophysiological measurement failed to reveal the presence of NK-lysin promoted channels [31]. Unlike NK-lysin, the net charge of the granulysin surface is exceptionally positive (11+), and these cationic residues are distributed over the entire surface of the molecule. There are no notable hydrophobic regions present in granulysin; only 11.5% of its surface is occupied by hydrophobic residues [16]. This is probably the reason that granulysin binds to lipopolysaccharide (LPS) and negatively charged phospholipid head groups in bacterial membranes, resulting in interference with the highly ordered lipid arrangement. Because of its di¡use charge distribution, it is di¤cult to predict the nature of the interaction of the protein with the lipid head groups. After binding to the membrane, granulysin or NKlysin may polymerize in the target cell membrane forming pores that lead to cell lysis. The mature form of NK-lysin is 78 amino acyl residues in length. NKG5 is one of the polymorphic forms of granulysin
[32]. Polymorphism also exists in porcine NK-lysin. Table 3 presents the NK-lysin proteins found in the database, while Fig. 3 shows their multiple sequence alignment. The residues in the K-helices are highly conserved, and all of the gaps are localized at the loop regions. The cysteines that characterize this family are marked with a dark background. 5. The pulmonary surfactant protein B (PSPB) family (Table 4 and Fig. 4) It has been 40 years since the ¢rst description of pulmonary surfactant was published [33] and more than 20 years since the ¢rst description of the socalled Sp-B protein appeared [34]. Pulmonary surfactant is a complex mixture of phospholipids and speci¢cally associated proteins. It reduces the surface tension at the air^liquid interfaces of the distal conducting airways and gas exchanging alveoli of the lung. Ninety percent of the surfactant complex is composed by lipids, and the remaining 10% is composed of at least three surfactant speci¢c proteins: Sp-A, Sp-B and Sp-C [35]. An Sp-B de¢ciency results from an inherited disease in full-term newly born infants which leads to lethal respiratory failure within the ¢rst year of life [36]. The mature Sp-B protein has 79 residues. Table 4 lists the Sp-B proteins re-
Fig. 3. Alignment of the NK-lysin family proteins.
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Table 4 Protein of the PSPB family Length
Source
Organism
Accession number
GI number
Description
PSPB Mmu
377
house mouse
M. musculus
P50405
1709875
PSPB PSPB PSPB PSPB PSPB
369 381 363 79 374
rabbit human dog pig sheep
O. cuniculus H. sapiens Canis familiaris S. scrofa Ovis aries
I46531 P07988 P17129 P15782 AAF14195
2137024 131418 131416 131419 6492136
pulmonary surfactantassociated protein B precursor (SP-B) surfactant protein B SP-B SP-B SP-B (8 kDa protein) SP-B
Ocu Hsa Cfa Ssc Oar
trieved from the database using the BLAST program, and Fig. 4 shows their sequence alignment. As in the other homologous families of the amoebapore superfamily, the six conserved cysteines, which are highlighted with a dark background, form three intramolecular disul¢de bridges [37]. There is another conserved cysteine in Sp-B which may be responsible for an intermolecular disul¢de bridge that causes dimerization of the protein. Human Sp-B has a net charge of 7+. Since the 3D structure of the protein has not been solved, it is still unknown how these charged residues are clustered, or how the charged residues might be arranged relative to anionic phospholipid head groups when the protein associates with a membrane. However it can be suggested that the action of Sp-B on membranes includes (1) membrane binding, (2) membrane lysis, (3) membrane fusion, (4) promotion of lipid adsorption to air^liquid surface ¢lms, and (5) respreading of ¢lms from the collapsed phase [38].
6. The acid sphingomyelinase (Asm) family (Table 5 and Fig. 5) Table 5 lists the three Asm proteins obtained from the databases. They are derived from Homo sapiens [39], Mus musculus [40] and Caenorhabditis elegans [41]. Although Asms have their own SAP activators, namely Sap B and Sap D, they also contain homologous SAP-like domains. Fig. 5 shows the sequence alignment of the three proteins. The six cysteines are marked with a dark background. The SAP-like motif is localized to the N-termini of the proteins. De¢ciencies in Sap B, Sap D and Asm result in three types of lysosomal storage disorders (metachromatic leukodystrophy (ML) [42], Gaucher disease [43] and Niemann^Pick disease (NPD) [44], respectively). According to the properties of SAPs and other SAPlike proteins, the SAP-like domain in Asm should possess lipid-binding properties. Furthermore, it must possess sphingomyelinase-activator properties because the addition of an activator such as Sap B or Sap D is not a prerequisite for activity [45].
Fig. 4. Sequence alignment of the PSPB family proteins.
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Table 5 Proteins of the Asm family Length
Source
Organism
Accession number
Asm Hsa
629
human
H. sapiens
P17405
Asm Mmu Asm Cel
627 572
house mouse worm
M. musculus C. elegans
Q04519 CAA91493
7. The acyloxyacyl hydrolase (AOAH) family (Table 6 and Fig. 6) AOAH is a leukocyte enzyme that cleaves bacterial
GI number 114258
1351982 3881643
Description sphingomyelin phosphodiesterase precursor (Asm) Asm similar to sphingomyelin phosphodiesterase
LPS and many glycerolipids. It contains two subunits. (1) The large subunit contains the signature sequence Gly-X-Ser-X-Gly which is found in the active sites of many lipases [46], and (2) the small sub-
Fig. 5. Sequence alignment of the Asm family proteins.
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Table 6 Proteins of the AOAH family Aoah Hsa Aoah Ocu Aoah Mmu
Length
Source
Organism
Accession number
GI number
Description
575 575 574
human rabbit house mouse
H. sapiens O. cuniculus M. musculus
4502115 AAB81183 M. musculus
4502115 2529573 2529571
AOAH precursor AOAH precursor AOAH precursor
unit is homologous to SAPs, exhibiting the characteristic six cysteines (Fig. 6). Like plant aspartic proteases (APs) and Asms, AOAH small subunits are covalently linked to the large subunits. Several experiments [47] indicate that the SAP-like domain not only plays an important role in facilitating rec-
ognition by AOAH of its target LPS, it also plays important roles in intracellular targeting and the catalytic activity of the enzyme. Thus, the enzyme was not found in lysosomes when the SAP-like domain was fully or partially deleted [47].
Fig. 6. Sequence alignment of the AHOH family proteins.
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Table 7 The proteins of the AP family Length
Source
Organism
Accession number
GI number
De¢nition
ASP2 Ath ASP Bna ASP Cpe ASP Vun ASP Han ASP Ccal CYP2 Ccar ASP Hvu
506 506 513 513 509 509 509 508
thale cress rape cucumber/squash cowpea sun£ower Centaurea calcitrapa Cynara cardunculus barley
Arabidopsis thaliana Brassica napus Cucurbita pepo Vigna unguiculata Helianthus annuus C. calcitrapa C. cardunculus Hordeum vulgare
AAC17620 AAB03108 O04057 AAB03843 BAA76870 CAA70340 S49349 P42210
3157937 1326165 2811025 1420936 4589716 1665867 1076696 1168536
ASP1 Osa
509
rice
O. sativa
Q42456
2499819
CYP1 Ccar ASP1 Ath Sen Hhy
474 508 517
C. cardunculus A. thaliana H. hybrid cr
CAA48939 AAD29758 AAC34854
509163 4773885 3551952
ASP Les
506
C. cardunculus thale cress Hemerocallis hybrid cultivar tomato
AAB18280
951449
ASP2 Osa PREB Cca PREA Cca ASP Bol ASP Car
496 506 504 292 204
rice C. cardunculus C. cardunculus cauli£ower chickpea
Lycopersicon esculentum O. sativa C. cardunculus C. cardunculus B. oleracea Cicer arietinum
AP precursor AP precursor AP precursor AP precursor AP precursor AP precursor cyprosin-cardoon phytepsin precursor (AP) AP oryzasin 1 precursor cyprosin putative AP senescenceassociated protein 4 AP precursor
P42211 CAB40349 CAB40134 CAA54478 BAA76427
1168537 4582534 4581209 459426 4586590
8. The aspartic protease (AP) family (Table 7 and Fig. 7) APs represent one of the four superfamilies of proteolytic enzymes. They are found in animals, plants, fungi, yeast, bacteria and viruses [48]. Plant APs contain an internal region consisting of about 100 residues which is not present in animal or microbial APs. This region is called `plant-speci¢c insert'. Its sequence and structure are quite similar to those of other SAP-like proteins except that its K-helical domains are interchanged, i.e., K-helices 1, 2, 3, 4 and 5 in SAPLIP are equivalent to K-helices 4, 5, 1, 2 and 3, respectively, in SAP. The evolutionary origin and the biological importance of this unique helix reshuf£ing event is obscure. It is likely that the plant-speci¢c domain plays an important role in vacuolar targeting. As for SAP and SAP-like proteins, the domain may bring the plant APs into contact with membranes and possibly also with a membranebound receptor in the Golgi apparatus. The resulting complex may then be packed into vesicles that transport APs into the vacuoles.
AP precursor preprocardosin B preprocardosin A AP precursor AP precursor
APs are activated by proteolytic cleavage, and the SAP-like domains are removed. Proteolysis may therefore destroy interaction with the putative membrane receptor or the membrane itself. This hypothesis has been substantiated with several lines of direct and indirect experimental evidence [10,49,50]. 9. The countin family Countin represents a new family within the amoebapore superfamily. Countin is a protein from Dictyostelium discoideum of 257 amino acyl residues [12]. From the sequence analyses we note that it also has the six cysteines of the amoebapore superfamily. Countin usually behaves as a complex of polypeptides with an e¡ective molecular mass of 450 kDa. It is able to `count' cell numbers and thereby regulate aggregate size. A cell aggregate breaks up into groups of smaller aggregates which thus form small fruiting bodies in response to starvation. The presence of the six-cysteine containing domain of the protein implies the presence of three disul¢de bridges
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Fig. 7. Sequence alignment of the AP family proteins.
and a tertiary structure similar to that of other members of the superfamily. We suggest that the other domains may play important roles in the `counting' function just as SAP-like domains in plant APs and
AOAHs serve related functions. Countin could interact with the cell membrane; then it might act as an activator to enhance the functions of other proteins in the membrane or merely activate other domains in C
Fig. 8. Sequence alignment of representative proteins within all of the families of the amoebapore superfamily except the AP family. Residues in bold print display the conserved cysteines while shaded residues for the NKL2 protein of S. cerevisiae show the transmembrane regions. The asterisk at the bottom of the alignment indicate the fully conserved cysteines.
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Table 8 Binary sequence comparisons which establish that proteins of eight families are members of a single superfamily Protein 1
Protein 2
Number of residues compared
Identity (%)
Similarity (%)
Comparison score (S.D.)
Sap Ddi PSPB Mmu Aoah Hsa Coun Ddi Asp2 Ath PSPB Mmu Asm Hsa
PFPB Ehi NKL2 Ssc Sap Bta PFPB Ehi PSPB Mmu PFPB Ehi Sap Hsa
139 78 77 102 95 98 83
28 24 31 29 21 23 27
41 41 46 41 36 37 34
15 12 12 11 11 11 10
the countin polypeptide chain. Facilitation of molecular interactions with membranes is a general characteristic of the proteins/domains of the amoebapore superfamily. 10. Conclusions Table 8 summarizes the statistical analyses of binary sequence comparisons obtained using the GAP program [13] with 500 random shu¥es. The comparison scores allow us to conclude that these proteins share a common evolutionary origin, and are therefore all members of a single diverse superfamily. The countin protein gives a comparison score of 11.4 S.D. with the PFPB Ehi protein. The newly identi¢ed protein therefore de¢nes a novel family within the amoebapore superfamily. Fig. 8 shows the sequence alignment of representative proteins from all of the families mentioned above, generated with the ClustalX program [51]. The plant AP family is not included since the Cand N-terminal regions are swapped. The conserved cysteines are displayed in bold font. The K-helical regions (according to the NMR structure of NK-lysin) are shaded in the NKL2 protein. By comparing the marked K-helical regions and the sequence alignment, we note that almost all of the gaps are in loop regions between the helices. Few gaps exist at the termini of the helices. This implies that all of the structurally unsolved proteins have similar K-helical structures and possibly related functions as well. Among the eight families discussed in this paper, only the amoebapore family has been shown by experiment to be able to form ion channels. Three families include enzymes with SAP-like domains.
These domains probably lead enzymes to their targets and enhance enzymatic activity. The disul¢de bridges in all amoebapore superfamily members render these proteins and protein domains remarkably stable to heat, acid and other environmental conditions. Their reduction reduces the antibacterial and cytolytic activities of NK-lysin, abolishes the pore forming activities of amoebapores and decreases the capacity of SAP to stimulate sphingolipid hydrolysis. Although proteins of the amoebapore superfamily di¡er widely in function, they have common properties related to lipid interactions. Countin is a newly identi¢ed member of the amoebapore superfamily. Its unique function in determining aggregate size during fruiting body formation represents a marked apparent deviation in function for a protein possessing a SAP-like domain. The presence of such a domain in countin suggests that its function is somehow related to the ability of the protein to bind to or otherwise recognize hydrophobic surfaces. Further experimentation will be required to reveal the molecular mechanism of action of this interesting protein.
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[29] N. Azuma, J. O'Brien, H.W. Moser, Y. Kishimoto, Arch. Biochem. Biophys. 311 (1994) 354^357. [30] M. Andersson, H. Gunne, B. Agerberth, A. Boman, T. Bergman, R. Sillard, H. Jornvall, V. Mutt, B. Olsson, W. Wigzell, EMBO J. 14 (1995) 1615^1625. [31] J.M. Ruysschaert, E. Goormaghtigh, F. Homble, M. Andersson, E. Liepinsh, G. Otting, FEBS Lett. 425 (1998) 341^344. [32] T. Yabe, C. McSherry, F.H. Bach, J.P. Houchins, J. Exp. Med. 172 (1990) 1159^1163. [33] J.A. Clements, Proc. Soc. Exp. Biol. Med. 95 (1957) 170^ 172. [34] P.J. Phizackerley, M.H. Town, G.E. Newman, Biochem. J. 183 (1979) 731^736. [35] G.S. Pryhuber, Mol. Genet. Metab. 64 (1998) 217^228. [36] A. Hamvas, L.M. Nogee, D.E. deMello, F.S. Cole, Biol. Neonate 67 (suppl 1) (1995) 18^31. [37] J. Johansson, T. Curstedt, H. Jo«rnuall, Biochemistry 30 (1991) 6917^6921. [38] S. Hawgood, M. Derrick, F. Poulain, Biochim. Biophys. Acta 1408 (1998) 150^160. [39] E.H. Schuchman, M. Suchi, T. Takahashi, K. Sandho¡, R.J. Desnick, J. Biol. Chem. 266 (1991) 8531^8539. [40] D. Newrzella, W. Sto¡el, Biol. Chem. Hoppe Seyler 373 (1992) 1233^1238. [41] The C. elegansSequencing Consortium,, Science 282 (1998) 2012^2018. [42] H. Holtschmidt, K. Sandho¡, H.Y. Kown, K. Harzer, T. Nakano, K. Suzuki, J. Biol. Chem. 266 (1991) 7556^7560. [43] D. Schnabel, M. Schro«der, K. Sandho¡, FEBS Lett. 284 (1991) 57^59. [44] E.H. Kolodny, Curr. Opin. Hematol. 7 (2000) 48^52. [45] S. Morimoto, B.M. Martin, Y. Kishimoto, J.S. O'Brien, Biochem. Biophys. Res. Commun. 156 (1988) 403^410. [46] B. Persson, G. Bengtsson-Olivecrona, S. Enerback, T. Olivecrona, H. Jornvall, Eur. J. Biochem. 179 (1989) 39^45. [47] J.F. Staab, D.L. Ginkel, G.B. Rosenberg, R.S. Munford, J. Biol. Chem. 269 (1994) 23736^23742. [48] N.D. Rawlings, A.J. Barrett, Methods Enzymol. 248 (1995) 105^120. [49] S. Glathe, J. Kervinen, M. Nimtz, G.H. Li, G.J. Tobin, T.D. Copeland, D.A. Ashford, A. Wlodawer, J. Costa, J. Biol. Chem. 273 (1998) 31230^31236. [50] T. Nakano, S. Murakami, T. Shoji, S. Yoshida, Y. Yamada, Plant Cell 9 (1997) 1653^1663. [51] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. Higgins, Nucleic Acids Res. 25 (1997) 4876^4882.
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The amoebapore superfamily Yufeng Zhai, Milton H. Saier Jr. * Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA Received 28 April 2000; accepted 8 June 2000
Abstract Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Amoebapore; Saposin; NK-lysin; Countin; Protozoan; Membrane; Pore; Disease; Molecular phylogeny
1. Introduction Amoebiasis is a disease caused by the protozoan parasite Entamoeba histolytica. The most prominent pathogenic feature of this disease is the powerful ability of the protozoan to kill target cells, resulting in massive tissue destruction [1]. Three amoebapore isoforms termed amoebapores A, B and C have been isolated. They form ion channels in membranes by oligomerization, and thus a¡ect membrane function and eventually result in lysis of the target cell [2]. Based on sequence and structural similarities, amoebapores are homologous to many other proteins, including saposins (SAPS) A, B, C and D [3], T-cell cytolytic proteins such as NK-lysins [4] and granuly-
* Corresponding author. Fax: 1-858-534-7108; E-mail: [email protected]
sins [5], pulmonary surfactant-associated proteins [6], acid sphingomyelinases (ASMs) [7,8], acyloxyacyl hydrolases (AOAH) [9], and swaposins which exhibit the SAP motif in plant aspartic proteases (AP) but with swapped N-terminal and C-terminal halves of the motif [10]. All of the proteins mentioned above and their homologous domains are termed saposinlike proteins, since SAPs A^D were the ¢rst proteins of this family to be characterized. Most of the proteins which comprise the amoebapore superfamily contain six conserved cysteine residues which form three disul¢de bridges. The NKlysins which contain ¢ve cysteines that form two disul¢de bridges represent the only exceptions to this structural pattern. These disul¢de bridges are in part responsible for the extraordinary thermostability of these proteins. For example, amoebapore A retains its activity after heating at 100³C for 15 min under non-reducing conditions [11].
0304-4157 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 5 7 ( 0 0 ) 0 0 0 0 3 - 4
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Interestingly, there is another protein, countin, which we here show to be a new member of the amoebapore superfamily. It is a newly characterized protein [12] which allows groups of slime mold cells to recognize their numbers and thereby regulate aggregate size. Like most other members of the amoebapore superfamily, countin contains a saposin-like domain with six conserved cysteine residues. In this paper we identify all currently sequenced members of the amoebapore superfamily, subdivide them into eight families (including the newly identi¢ed `countin' family), present the sequence alignments, and discuss the structures and functions of these proteins. We provide evidence that all these proteins including countin are evolutionarily related to each other. 2. The pore-forming amoebapore (PFA) family (Table 1 and Fig. 1) Amoebapores, pore forming peptides of the protozoan E. histolytica, are the cause of tissue destruction in the human host. Experimental data indicate that amoebapores create ion channels in membrane by oligomerization [2]. Three isoforms, termed amoebapore A, B and C, with mature proteins of 77-residues in length, exist in cytoplasmic granules of the amoeba. They display antibacterial activity and are instrumental in preventing the growth of phagocitized bacteria. Secondary structural predictions suggested that
amoebapore has four amphipathic alpha-helical domains [14] stabilized by six cysteines which form three disul¢de bridges (Fig. 1). In Fig. 1 and in subsequent ¢gures in this paper, the sequences of all or most members of a family are aligned; however, if two or more homologues show very high percent identities, only one of them is shown. The 3D structure of NK-lysin has been solved by NMR [15], and the structure of amoebapore has been postulated based on homology modeling methods. The theoretical structure, which has ¢ve K-helices arranged in two layers, provides insight into possible structure^function relationships [16]. The results show that amoebapores A, B and C exhibit hydrophobic accessible surface areas that correspond to about 26, 32 and 32% of the total surface, respectively. The largest hydrophobic areas overlap helices 1, 3 and 5, creating hydrophobic grooves between the two layers. The hydrophobic cores between the two layers are loosely packed according to cavity analysis. Besides these features, there is no biased charge distribution in any of the isoforms. This feature differs from that suggested for NK-lysin, implying a di¡erent mechanism of membrane interaction. The hypothesized mechanism suggests a conformational rearrangement and concomitant membrane insertion of helices 2 and 3 after initial lipid binding and oligomerization. The resultant transmembrane pore comprises a permeability barrier and may eventually lyse the target cell. All of the nearest amoebapore subfamily homo-
Table 1 Proteins of the PFA family Source organism
Accession number
GI number
Database description
PFPB Ehi
Length 96
E. histolytica
Q24824
2498767
PFPB Edi
96
E. dispar
AAF04195
6119727
PFPA Ehi
98
E. histolytica
P34095
464366
PFPN Ehi
97
E. histolytica
Q07831
730303
PFPC1 Ehi PFPC2 Ehi
88 101
E. histolytica E. histolytica
BAA22028 Q24825
2351011 2498768
PFPC Edi
101
E. dispar
AAF04196
6119729
pore-forming peptide amoebapore B precursor pore-forming protein isoform B precursor pore-forming peptide amoebapore A precursor nonpathogenic pore-forming peptide precursor amoebapore C pore-forming peptide amoebapore C precursor pore-forming protein isoform C precursor
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Fig. 1. Sequence alignment of pore-forming amoebapore family proteins. In this and the subsequent ¢gures, protein abbreviations are as indicated in Table 1 or in the corresponding table. Shaded residues are the conserved cysteines. An asterisk below the alignment indicates a fully conserved residue, while one or two dots indicate distant and close similarities, respectively.
logues retrieved from the database using the BLAST [17] search tool are presented in Table 1. These proteins are from Entamoeba histolytica and E. dispar. The two homologues obtained from E. dispar are closely related to those from E. histolytica. Both species live as commensals in the lower human intestine. However only the latter causes disease [18]. In vitro, both amoeba can kill host cells, but E. dispar is approximately threefold less e¡ective than E. histolytica [19]. 3. The saposin (SAP) family (Table 2 and Fig. 2) The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called SAPs which are generated from common precursors called prosaposins [20]. Selective SAP de¢ciencies are known only for Sap B and C. A de¢ciency in Sap B or C causes increased storage of sulfatides [21,22] and glucosylcermides [23]. De¢ciencies of Sap A or D have not been reported. Although their physiological functions have not been ¢rmly established, one hypothesis is that Sap A stimulates the hydrolysis of glucosyl- and galactosylceramides [24,25]. Sap D is likely to stimulate degradation of ceramides since its addition to the culture medium of ¢broblasts from patients with a prosaposin de¢ciency led to a decrease in accumulated ceramide [26]. Mature SAPs are small proteins of about 80 amino acids that share several structural properties which include the six conserved cysteines. Although their involvement in the degradation of sphingolipids is
well established, their mechanisms of action are still under debate [27]. It has been assumed that the mode of action of Sap B is based on its capacity to solubilize sulfatides to make them accessible to the degradative action of arylsulfatases. As to Sap C, the physiological activator of glucosylceramidase, it is thought that it enhances enzyme activity by forming an active complex with the enzyme [20]. Recently this hypothesis has been questioned. Sap C may instead promote glucosylceramidase localization to lipid surfaces [28]. Sap A also stimulates the glucosylceramidase activity probably by inducing a conformational change in the enzyme. However, detailed information regarding its mechanism of action is lacking. It was hypothesized that the action of Sap D depends on its interaction with the enzyme rather than with the ceramide substrate [29]. Table 2 and Fig. 2 present a list of the SAP proteins retrieved from the databases and the alignment of their sequences, respectively. 4. The NK-lysin/granulysin family (Table 3 and Fig. 3) NK-lysin and granulysin are anti-bacterial peptides produced by pig natural killer cells [30] and human cytolytic lymphocytes [5], respectively. NKlysin shows antibacterial activity as well as cytolytic activity toward certain cancer cells. Granulysin exhibits a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. Multiple sequence alignments show that
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Table 2 Proteins of the SAP family Length
Source
Organism
Accession number
GI number
Description
pig
Sus scrofa
P81405
3914938
slime mould cow
D. discoideum Bos taurus
BAA32237 P26779
3402901 134216
guinea pig
Cavia porcellus
P20097
134217
524
human
H. sapiens
P07602
134218
518 554 554 556 402
chicken rat house mouse mouse worm
Gallus gallus Rattus sp. M. musculus Mus sp. C. elegans
AAF05899 AAB36233 AAA92567 AAB31059 AAB37548
Sap B (cerebroside sulfate activator) Sap A Sap C (co-L-glucosidase) Sap C (co-L-glucosidase) proactivator polypeptide precursor prosaposin prosaposin sulfated glycoprotein prosaposin weak similarity to mouse sphingolipid activator protein
Sap Ssc
79
Sap Ddi Sap Bta
143 80
Sap Cpo
81
Sap Hsa Sap Sap Sap Sap Sap
Gga Rsp Mmu Msp Cel
these proteins have invariant cysteine residues as do the SAPs and amoebapores, but the ¢rst cysteine in granulysin is replaced by tyrosine as shown in Fig. 3 which allows it to form only two disul¢de bridges. This di¡erence is thought to be of little signi¢cance with respect to protein stability [16]. The 3D structure of NK-lysin, recently solved by NMR spectroscopy, provides a template for estimation of the structures of other SAP-like proteins such as granulysin and amoebapore [15]. There is a groove
6224674 1336844 881390 557967 1065921
at the interface of helices 1, 3 and 5, and the protein exhibits small hydrophobic patches near the C-terminal surface of helix 1. Hydrophobic residues occupy about 17% of the accessible surface of this protein. NK-lysin has a net charge of 6+, and most of these charges are arranged in an equatorial belt around the molecule in a vertical position of helix 1. This belt divides a more negatively charged half of the molecule from the other half around the N- and C-termini which contain few acidic residues. These character-
Fig. 2. Sequence alignment of SAP family proteins.
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Table 3 Proteins of the NK-lysin family TCAP Hsa Gran Hsa NKG5 Hsa NKL1 Ssc NKL2 Ssc
Length
Source
Organism
Accession number
GI number
Description
129 145 145 129 78
human human human pig pig
H. sapiens H. sapiens H. sapiens S. scrofa S. scrofa
A27562 NP_006424 CAA38035 Q29075 2392473
88664 5453784 35065 2498644 2392473
T-cell activation protein granulysin NKG5 product NK-lysin precursor NK-Lysin
istics of NK-lysin imply a possible mechanism of membrane interaction. The N-terminal and C terminal residues, localized to helices 1 and 5, respectively, dip into the membrane; the positively charged belt interacts with the phospholipid head groups, and the other half of the protein is exposed to the solvent. The potential for pore formation remains unclear, and electrophysiological measurement failed to reveal the presence of NK-lysin promoted channels [31]. Unlike NK-lysin, the net charge of the granulysin surface is exceptionally positive (11+), and these cationic residues are distributed over the entire surface of the molecule. There are no notable hydrophobic regions present in granulysin; only 11.5% of its surface is occupied by hydrophobic residues [16]. This is probably the reason that granulysin binds to lipopolysaccharide (LPS) and negatively charged phospholipid head groups in bacterial membranes, resulting in interference with the highly ordered lipid arrangement. Because of its di¡use charge distribution, it is di¤cult to predict the nature of the interaction of the protein with the lipid head groups. After binding to the membrane, granulysin or NKlysin may polymerize in the target cell membrane forming pores that lead to cell lysis. The mature form of NK-lysin is 78 amino acyl residues in length. NKG5 is one of the polymorphic forms of granulysin
[32]. Polymorphism also exists in porcine NK-lysin. Table 3 presents the NK-lysin proteins found in the database, while Fig. 3 shows their multiple sequence alignment. The residues in the K-helices are highly conserved, and all of the gaps are localized at the loop regions. The cysteines that characterize this family are marked with a dark background. 5. The pulmonary surfactant protein B (PSPB) family (Table 4 and Fig. 4) It has been 40 years since the ¢rst description of pulmonary surfactant was published [33] and more than 20 years since the ¢rst description of the socalled Sp-B protein appeared [34]. Pulmonary surfactant is a complex mixture of phospholipids and speci¢cally associated proteins. It reduces the surface tension at the air^liquid interfaces of the distal conducting airways and gas exchanging alveoli of the lung. Ninety percent of the surfactant complex is composed by lipids, and the remaining 10% is composed of at least three surfactant speci¢c proteins: Sp-A, Sp-B and Sp-C [35]. An Sp-B de¢ciency results from an inherited disease in full-term newly born infants which leads to lethal respiratory failure within the ¢rst year of life [36]. The mature Sp-B protein has 79 residues. Table 4 lists the Sp-B proteins re-
Fig. 3. Alignment of the NK-lysin family proteins.
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Table 4 Protein of the PSPB family Length
Source
Organism
Accession number
GI number
Description
PSPB Mmu
377
house mouse
M. musculus
P50405
1709875
PSPB PSPB PSPB PSPB PSPB
369 381 363 79 374
rabbit human dog pig sheep
O. cuniculus H. sapiens Canis familiaris S. scrofa Ovis aries
I46531 P07988 P17129 P15782 AAF14195
2137024 131418 131416 131419 6492136
pulmonary surfactantassociated protein B precursor (SP-B) surfactant protein B SP-B SP-B SP-B (8 kDa protein) SP-B
Ocu Hsa Cfa Ssc Oar
trieved from the database using the BLAST program, and Fig. 4 shows their sequence alignment. As in the other homologous families of the amoebapore superfamily, the six conserved cysteines, which are highlighted with a dark background, form three intramolecular disul¢de bridges [37]. There is another conserved cysteine in Sp-B which may be responsible for an intermolecular disul¢de bridge that causes dimerization of the protein. Human Sp-B has a net charge of 7+. Since the 3D structure of the protein has not been solved, it is still unknown how these charged residues are clustered, or how the charged residues might be arranged relative to anionic phospholipid head groups when the protein associates with a membrane. However it can be suggested that the action of Sp-B on membranes includes (1) membrane binding, (2) membrane lysis, (3) membrane fusion, (4) promotion of lipid adsorption to air^liquid surface ¢lms, and (5) respreading of ¢lms from the collapsed phase [38].
6. The acid sphingomyelinase (Asm) family (Table 5 and Fig. 5) Table 5 lists the three Asm proteins obtained from the databases. They are derived from Homo sapiens [39], Mus musculus [40] and Caenorhabditis elegans [41]. Although Asms have their own SAP activators, namely Sap B and Sap D, they also contain homologous SAP-like domains. Fig. 5 shows the sequence alignment of the three proteins. The six cysteines are marked with a dark background. The SAP-like motif is localized to the N-termini of the proteins. De¢ciencies in Sap B, Sap D and Asm result in three types of lysosomal storage disorders (metachromatic leukodystrophy (ML) [42], Gaucher disease [43] and Niemann^Pick disease (NPD) [44], respectively). According to the properties of SAPs and other SAPlike proteins, the SAP-like domain in Asm should possess lipid-binding properties. Furthermore, it must possess sphingomyelinase-activator properties because the addition of an activator such as Sap B or Sap D is not a prerequisite for activity [45].
Fig. 4. Sequence alignment of the PSPB family proteins.
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Table 5 Proteins of the Asm family Length
Source
Organism
Accession number
Asm Hsa
629
human
H. sapiens
P17405
Asm Mmu Asm Cel
627 572
house mouse worm
M. musculus C. elegans
Q04519 CAA91493
7. The acyloxyacyl hydrolase (AOAH) family (Table 6 and Fig. 6) AOAH is a leukocyte enzyme that cleaves bacterial
GI number 114258
1351982 3881643
Description sphingomyelin phosphodiesterase precursor (Asm) Asm similar to sphingomyelin phosphodiesterase
LPS and many glycerolipids. It contains two subunits. (1) The large subunit contains the signature sequence Gly-X-Ser-X-Gly which is found in the active sites of many lipases [46], and (2) the small sub-
Fig. 5. Sequence alignment of the Asm family proteins.
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Table 6 Proteins of the AOAH family Aoah Hsa Aoah Ocu Aoah Mmu
Length
Source
Organism
Accession number
GI number
Description
575 575 574
human rabbit house mouse
H. sapiens O. cuniculus M. musculus
4502115 AAB81183 M. musculus
4502115 2529573 2529571
AOAH precursor AOAH precursor AOAH precursor
unit is homologous to SAPs, exhibiting the characteristic six cysteines (Fig. 6). Like plant aspartic proteases (APs) and Asms, AOAH small subunits are covalently linked to the large subunits. Several experiments [47] indicate that the SAP-like domain not only plays an important role in facilitating rec-
ognition by AOAH of its target LPS, it also plays important roles in intracellular targeting and the catalytic activity of the enzyme. Thus, the enzyme was not found in lysosomes when the SAP-like domain was fully or partially deleted [47].
Fig. 6. Sequence alignment of the AHOH family proteins.
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Table 7 The proteins of the AP family Length
Source
Organism
Accession number
GI number
De¢nition
ASP2 Ath ASP Bna ASP Cpe ASP Vun ASP Han ASP Ccal CYP2 Ccar ASP Hvu
506 506 513 513 509 509 509 508
thale cress rape cucumber/squash cowpea sun£ower Centaurea calcitrapa Cynara cardunculus barley
Arabidopsis thaliana Brassica napus Cucurbita pepo Vigna unguiculata Helianthus annuus C. calcitrapa C. cardunculus Hordeum vulgare
AAC17620 AAB03108 O04057 AAB03843 BAA76870 CAA70340 S49349 P42210
3157937 1326165 2811025 1420936 4589716 1665867 1076696 1168536
ASP1 Osa
509
rice
O. sativa
Q42456
2499819
CYP1 Ccar ASP1 Ath Sen Hhy
474 508 517
C. cardunculus A. thaliana H. hybrid cr
CAA48939 AAD29758 AAC34854
509163 4773885 3551952
ASP Les
506
C. cardunculus thale cress Hemerocallis hybrid cultivar tomato
AAB18280
951449
ASP2 Osa PREB Cca PREA Cca ASP Bol ASP Car
496 506 504 292 204
rice C. cardunculus C. cardunculus cauli£ower chickpea
Lycopersicon esculentum O. sativa C. cardunculus C. cardunculus B. oleracea Cicer arietinum
AP precursor AP precursor AP precursor AP precursor AP precursor AP precursor cyprosin-cardoon phytepsin precursor (AP) AP oryzasin 1 precursor cyprosin putative AP senescenceassociated protein 4 AP precursor
P42211 CAB40349 CAB40134 CAA54478 BAA76427
1168537 4582534 4581209 459426 4586590
8. The aspartic protease (AP) family (Table 7 and Fig. 7) APs represent one of the four superfamilies of proteolytic enzymes. They are found in animals, plants, fungi, yeast, bacteria and viruses [48]. Plant APs contain an internal region consisting of about 100 residues which is not present in animal or microbial APs. This region is called `plant-speci¢c insert'. Its sequence and structure are quite similar to those of other SAP-like proteins except that its K-helical domains are interchanged, i.e., K-helices 1, 2, 3, 4 and 5 in SAPLIP are equivalent to K-helices 4, 5, 1, 2 and 3, respectively, in SAP. The evolutionary origin and the biological importance of this unique helix reshuf£ing event is obscure. It is likely that the plant-speci¢c domain plays an important role in vacuolar targeting. As for SAP and SAP-like proteins, the domain may bring the plant APs into contact with membranes and possibly also with a membranebound receptor in the Golgi apparatus. The resulting complex may then be packed into vesicles that transport APs into the vacuoles.
AP precursor preprocardosin B preprocardosin A AP precursor AP precursor
APs are activated by proteolytic cleavage, and the SAP-like domains are removed. Proteolysis may therefore destroy interaction with the putative membrane receptor or the membrane itself. This hypothesis has been substantiated with several lines of direct and indirect experimental evidence [10,49,50]. 9. The countin family Countin represents a new family within the amoebapore superfamily. Countin is a protein from Dictyostelium discoideum of 257 amino acyl residues [12]. From the sequence analyses we note that it also has the six cysteines of the amoebapore superfamily. Countin usually behaves as a complex of polypeptides with an e¡ective molecular mass of 450 kDa. It is able to `count' cell numbers and thereby regulate aggregate size. A cell aggregate breaks up into groups of smaller aggregates which thus form small fruiting bodies in response to starvation. The presence of the six-cysteine containing domain of the protein implies the presence of three disul¢de bridges
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Fig. 7. Sequence alignment of the AP family proteins.
and a tertiary structure similar to that of other members of the superfamily. We suggest that the other domains may play important roles in the `counting' function just as SAP-like domains in plant APs and
AOAHs serve related functions. Countin could interact with the cell membrane; then it might act as an activator to enhance the functions of other proteins in the membrane or merely activate other domains in C
Fig. 8. Sequence alignment of representative proteins within all of the families of the amoebapore superfamily except the AP family. Residues in bold print display the conserved cysteines while shaded residues for the NKL2 protein of S. cerevisiae show the transmembrane regions. The asterisk at the bottom of the alignment indicate the fully conserved cysteines.
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98
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Table 8 Binary sequence comparisons which establish that proteins of eight families are members of a single superfamily Protein 1
Protein 2
Number of residues compared
Identity (%)
Similarity (%)
Comparison score (S.D.)
Sap Ddi PSPB Mmu Aoah Hsa Coun Ddi Asp2 Ath PSPB Mmu Asm Hsa
PFPB Ehi NKL2 Ssc Sap Bta PFPB Ehi PSPB Mmu PFPB Ehi Sap Hsa
139 78 77 102 95 98 83
28 24 31 29 21 23 27
41 41 46 41 36 37 34
15 12 12 11 11 11 10
the countin polypeptide chain. Facilitation of molecular interactions with membranes is a general characteristic of the proteins/domains of the amoebapore superfamily. 10. Conclusions Table 8 summarizes the statistical analyses of binary sequence comparisons obtained using the GAP program [13] with 500 random shu¥es. The comparison scores allow us to conclude that these proteins share a common evolutionary origin, and are therefore all members of a single diverse superfamily. The countin protein gives a comparison score of 11.4 S.D. with the PFPB Ehi protein. The newly identi¢ed protein therefore de¢nes a novel family within the amoebapore superfamily. Fig. 8 shows the sequence alignment of representative proteins from all of the families mentioned above, generated with the ClustalX program [51]. The plant AP family is not included since the Cand N-terminal regions are swapped. The conserved cysteines are displayed in bold font. The K-helical regions (according to the NMR structure of NK-lysin) are shaded in the NKL2 protein. By comparing the marked K-helical regions and the sequence alignment, we note that almost all of the gaps are in loop regions between the helices. Few gaps exist at the termini of the helices. This implies that all of the structurally unsolved proteins have similar K-helical structures and possibly related functions as well. Among the eight families discussed in this paper, only the amoebapore family has been shown by experiment to be able to form ion channels. Three families include enzymes with SAP-like domains.
These domains probably lead enzymes to their targets and enhance enzymatic activity. The disul¢de bridges in all amoebapore superfamily members render these proteins and protein domains remarkably stable to heat, acid and other environmental conditions. Their reduction reduces the antibacterial and cytolytic activities of NK-lysin, abolishes the pore forming activities of amoebapores and decreases the capacity of SAP to stimulate sphingolipid hydrolysis. Although proteins of the amoebapore superfamily di¡er widely in function, they have common properties related to lipid interactions. Countin is a newly identi¢ed member of the amoebapore superfamily. Its unique function in determining aggregate size during fruiting body formation represents a marked apparent deviation in function for a protein possessing a SAP-like domain. The presence of such a domain in countin suggests that its function is somehow related to the ability of the protein to bind to or otherwise recognize hydrophobic surfaces. Further experimentation will be required to reveal the molecular mechanism of action of this interesting protein.
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