Akira SanoS, Norman S. RadinSQ, Linda L. Johnsonll, and George E. Tarry. From the $ Mental ..... Basu, A., Glew, R. H., Daniels, L. B., and Clark, L. S. (1984) J. Fujibayashi, S., Kao .... IlYerS from Pel-Freez Biologicals (Rogers. AZ) were also ...
Vol. 263,No. 36. Issue of December 25, pp. 19597-19601,1988 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 I988 by TheAmerican Society far Biochemistry and Molecular Biology, Inc.
The Activator Protein forGlucosylceramide ,&Glucosidase from Guinea Pig Liver IMPROVED ISOLATION METHOD AND COMPLETE AMINOACID
SEQUENCE* (Received for publication, May 2, 1988)
Akira SanoS, Norman S. RadinSQ, Linda L. Johnsonll, and George E. Tarry From the $ Mental Health Research Institute andthe YDepartment of Biological Chemistry, the University of Michigan, Ann Arbor, Michigan 48109
&Glucosidase activator (SAP-2) is a family of heat- cosidase in a wide variety of organisms and cells to form stable, acidic glycoproteins which stimulate enzymatic ceramide and glucose. The enzyme activity in uitro is greatly hydrolysis of glucosylceramide. In this study, we im- stimulated by taurocholate, acidic lipids, or- in thepresence proved the purification method and found that SAP-2 of a small amount of acidic lipid-by an activator proteinthat is highly heterogeneous. A hot water extractof frozen has been called Factor P (l),HSF (2), SAP-2 (3), coglucosiguinea pig liver was fractionated by ammoniumsulfate sedimentation, then chromatographed withDEAE-Se- dase (4),cohydrolase sphingolipid I (5), AP (6), and sphinphacel, Sephadex G-75, and concanavalin A-Sepha- golipid activator protein A (7). For brevity here we call it rose. A fraction binding toconcanavalin A-Sepharose SAP-2. It has been said to stimulate also sphingomyelinase was purifiedfurther with a C4 high performance liquid and galactosylceramide P-galactosidase. The protein hasbeen chromatography reverse phase column. This yielded isolated from bovine andhuman spleen and from human several peaks, the main one of which was studied. The brain (4, 5, 8, 9). It showed heterogeneity in PAGE, and specific activity of the purifiedSAP-2 was 35 units/Mg concanavalin A chromatography yielded binding and non(1 unit produces 50% stimulation of a basalglucosidase binding forms of the protein (5, 6). The different members of preparation). N-terminal amino acid sequencing the family were found to have similar stimulating activities, showed that this preparation isa mixture of polypeptides differing in the presence or absence of one or two stainability on PAGE with Stains All, and immunoreactivity of the end amino acids. The complete amino acid se- with polyclonal antibodies prepared against the mixture (5). quence of the 81 residues in SAP-2 has been deter- SAP-2 accumulates greatly in the spleen of patients with mined. Comparison of the sequence of guinea pig SAP- Gaucher disease (genetic defect in glucosidase activity), and 2 with the sequence of human sphingomyelinase acti- its accumulation in liver could be induced by injecting emulvator revealed 58% homology and quite similar hy- sified GlcCer into mice (10). SAP-2 apparently acts by comdropathy profiles. Both proteins possess a highly hy- bining with the enzyme and acidic lipid to form an activated drophilic region around Asn-22, which is glycosylated, complex, rather than by simply solubilizing the substrate (11). and 6 cysteine residues, in oxidized form, located in The SAP-2s from normal and Gaucher human spleen were the samepositions. Comparison with thepublished nucleotide sequence for theprecursor formof the human found to differ slightly in molecular weight and electrophoactivator protein for sulfatide sulfatase (SAP-1) sug- retic properties, but all preparations seemed to work on engested that this activator also has a possibly glycosy- zymes from different sources. An activator preparation acting on sphingomyelinase and, lated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat to some extent, on GlcCer P-glucosidase, has been isolated different. Examination of another region of the pre- from a Gaucher spleen and its complete amino acid sequence cursor’s nucleotide sequence, assuming a few changes has been described (12). In this paper, we describe an imin the identifications, revealed the presence of the proved method for isolating several forms of glucosidase actisphingomyelinase activator. It appears that two or vators from a normal tissue, guinea pig liver, describe the more activators are derived from a single precursor primary structure of one form of the activator, and point to protein. Marked homologies were seen also with a lung the similarities and differences in the structures of the two surfactant protein and a sulfated glycoprotein from proteins. Previously published data on a precursor protein of Sertoli cells. a different activator are reinterpreted and some striking homologies with other proteins arenoted. The glycosphingolipid, GlcCer,’ is catabolized by a p-glu~
~
~~~
EXPERIMENTAL PROCEDURES A N D RESULTS~
* This study was supported by Grant NS 03192 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. fiRecipient of the Senator Jacob Javits Neuroscience Investigator Award from the National Institutes of Health. To whom correspondence and reprint requests should be addressed. The abbreviations used are: GlcCer, glucosylceramide or glucocerebroside; BSA, bovine serum albumin; Buffer A, 10 mM sodium phosphate, pH 7.0, 0.02% NaN3; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SAP-2, P-glucosidase-activatingprotein; SDS, sodium dodecyl sulfate; TFA, trlfluoracetic acid ConA, concanavalin A.
DISCUSSION
Comparison of Hydrolase Activators-Comparison in Fig. 5 of the sequences of SAP-2 and of AI. sphingomyelinase activator from human Gaucher spleen (12) shows that there is 58% homology and that the residues important for protein
* Portions of this paper (including “Experimental Procedures,” “Results,” Tables 1-111, and Figs. 1-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
19597
for
19598
Protein Activator
1 5 NH24lU-SOr-V.l-Thr-8-LyS"la-Cp.Olu-Tyr-
P-Glucosidase
10
NH2-SOr-Asp-V~-Tyr-Cy8-Glu-Val-Cy.Olu-Phe20
11 15 Val-V.l-Ly8-Lys-V~l-~et-Glu-L.u-Il.-A.pLeu-V8l-Ly8-Glu-Val-Thr-Lys-L.u-Ilo-A8p-
0
21
>,
A8ua-~E-Arg-Thr-Olu-Glu-Lys-Il."le-His-
A8un-UX-Lys-Thr-Glu-Lys-Glu-Ilo-Leu-Asp31
40
D1a-LeU-&p-Ser-Val-Cy.-Ala-Leu-L.u-ProAh-Phe-A8p-Lys-Met-Cy8-Ser-Lys-L.u-Pro50
41
z
-
15 10
C
5
5
0
a
-5
a
xe
-10
-
-15
G.pigSAP-2
45
Glu-Sor-Val-Sor-Glu-Val-Cya-Gln-Glu-ValLys-Sor-Leu-Sor-Glu-Glu-Cy.-Gln-Glu-Val51
Residue Number
Val-A8p-Thr-Tyr-Gly-Asp-Sor-Ilo-Val-AlaVal-A8p-Thr-Tyr-Gly-Ser-Sor-Ilo-Leu-Ser70
61
65
Leu-L.u-L.u-Gln-Glu-Met-S~r-Pro-Glu-L.uIle-L.u-L.u-Glu-Glu-Val-Sor-Pro-Glu-L.u71
-
80
Val-Cy8-Ser-Glu-L.u-Gly-L.u-Cy6-Met-Ser-Gly-COOH V8l-Cy8-Sor-Met-L.u-His-L.u-Cy8-Ser-Gly-COOH
FIG. 5. Amino acid sequence of guinea pig liver SAP-2 (upperline) and Gaucher spleen sphingomyelinase activator (lower line). The matching residues appear in bold print.
structure (Cys-5, -8, -36, -47, -72, and -78 and glycosylated Asn-22) are identical. The finally deduced sequence for the Ala protein was based on peptides which failed to overlap in three different points; nevertheless, the alignment is in good agreement with ours. One point of difference between the two is in the C-terminal region which, in SAP-2, ends with an 81st residue and in Ala ends in an 80thresidue. The differences can be attributed toevolutionary differentiation and, possibly, also to theexistence of more than one form of SAP-2 (7). The hydropathy profiles of the two activator proteins, calculated by the method of Kyte and Doolittle (24), were surprisingly similar despite the 42% residue substitutions (Fig. 6A). The first 30 and the last 30 residues were very similar while a significant difference in the overall hydropathy characteristics appeared in the central region. Despite this difference, the central regions showed remarkable similarity in the positions of the minor peaks and valleys. The oligosaccharide moiety, at Asn-22, is sited at the center of a somewhat hydrophilic segment of the protein, thus augmenting its hydrophilicity. Analysis of the nucleotide sequence of the cDNA corresponding to another human activator protein (SAP-1) has been reported (25). This protein stimulates the activity of other sphingolipid hydrolases. The sequence codes for a large precursor form of the activator and most of the polypeptide has no known function. The SAP-1 sequence is believed to comprise nucleotides 565-801 (79 amino acids). The sequence predicts the presence of 6 Cys residues at positions 4,7, 36, 47, 71, and 77, which differ from those of the Ala protein and SAP-2 in that the first two and last two are 1 residue lower. Asn is assigned position 21, also one lower than the glycosylated Asn in AI, and SAP-2. All three proteins are strikingly similar with respect to length, the positions of the Cys residues, and the glycosylation site. The hydropathy profiles of human SAPsl and2 are fairly similar, despite the low degree of homology (Fig. 6B). The former lacks the strong hydrophilic region around Asn-22, although it does exhibit the
15
Human SAP-1
X
-4*
10
C
5
5
0
e
-5
a a
-10
I -1 5
-20 -
2 0
5
t " 10
~ a 20
"
30
'
'
r 40
1
' ' 50
" ' 60
1 70
80
Residue Number FIG. 6. Comparison of hydropathy profilesof activator proteins. A segment width of 9 residues was used in the calculation. A, guinea pig SAP-2 and human AI..E , human SAP-1 and human Ala.
somewhat hydrophilic region in the center of the molecule. Another intriguing observation comes from reinterpretation of the nucleotide sequence for this precursor protein (25): ifwe omit adenylic acid 1019 from the data and then reencode the triplets starting with nucleotide 1018, we obtain the sequence for the human activator Ala up to amino acid residue 67 (nucleotide 1111).If we further suggest that nucleotides 1112 and 1113 should be replaced by three nucleotides coding for Pro, the remaining 12 amino acid residues of activator Ala are also in agreement with the nucleotide sequence. Thus, it seems highly likely that the large precursor protein codes for two different sphingolipid hydrolase activators. This conclusion is supported by the observations that human chromosome 10 codes for both activators (26, 27). Ifwe further examine the precursor DNA sequence and assume guanidylic acid 1192 is the beginning of a third activator protein( X ) ,it is then possible to see an Asn-22 followed by Ser-Thr (again a presumed glycosylated residue) and 5CYS residues in the same positions as in SAP-2 and AI.. Only the 6th Cys residue seems to be missing. All four proteins have a Tyr-54. The hydropathy profiles for X and Ala (not shown) are strikingly similar despite the low degree of homology. It is unlikely that this thirdregion produces the hexosaminidase activator for the enzyme that acts on ganglioside G w (28), since the activator is encoded on a different chromosome.
ProteinActivator Subsequent to this reinterpretation, we have learned (29) that the precursor protein for SAP-1 does indeed contain within it a very close copy of the SAP-2 protein, as well as two more very similar proteins (presumably other sphingolipid hydrolase activators). Structure-Function Correlation-The basic amino acids in guinea pig SAP-2 are located in the first 27 N-terminal residues; in thehuman activator,Ala, they arelocalized in the first 41 residues (Fig. 5). The acidic amino acids, on theother hand, are rather diffusely distributed. Thus, especially at the acidic pH characteristic of lysosomes, the glucosidase activators possess a distinctpositively charged region. Both proteins also possess a hydrophobic region due to the first 19 residues (Figs. 3 and 6 A ) .Thus, this region is a good candidate for the binding of an acidic lipid in the active ternary complex with the enzyme (11,30). The hydrophilic region of SAP-2 apparently is not involved in the binding to p-glucosidase since deglycosylation in this region did not destroy the stimulating activity (18). SAP-1, unlike the glucosidase activator, has been shown to form a solubilizing complex with the substrates of the sphingolipid hydrolases that it activates and apparently does not interact positively with acidic lipids. These differences in mechanism of action are consistentwith the above hypothesis since SAP-1 lacks a basic amino acid sequences. Possibly the hydrophobic regions due to residues 3-18 and 24-29 are responsible for the substrate binding (Fig. 6B). Dewji et al. (25) have pointed to the marked similarity in sequence pattern and homology between human SAP-1 and a region of a sulfated glycoprotein from rat Sertoli cells (31). The sequence homology between the two proteins was 76%. In viewof the close similarity, we suggest that the Sertoli protein functions as an activator (or inhibitor) for some of the hydrolases in the sperm acrosome, a region needed for penetration of the egg cell zona pellucida. A more recent paper on the Sertoli protein (32) has shown that it possesses four domains, very much like the sphingolipid activator precursor protein (29). In addition, it maybe noted that a lipid-binding lung surfactantprotein (33) also has the same patternasthe hydrolase activators, particularly the positions of the 6 Cys residues (oxidized), the Tyr following the second pair of Cys residues, and the 2 Pro residues near the third and fifth Cys groups. However, the lung protein lacks the glycosylation site, a feature which helps explain its extreme lipophilic nature. The overall homology between human Ala andsurfactant protein amounts to 20 amino acids out of 80. More recent work on this protein3 has shown that it too occurs as part of a precursor protein with more than three similar domains, like the SAP proteins. Acknowledgments-We are indebted to Dr. Fulvio Perini and Laura L.Loesel of the Department of Pharmacology for assistance in sequence analysis. The Protein Identification Resource, Georgetown University Medical Center, kindly used their database and computer to find the marked similarity between SAP-2 and surfactant proteins, as well as additional intriguing relationships. Inez Mason contributed valuable laboratory assistance. Note added in Proof-Furst et al. (34) have also recently noted the presence of SAP-1 andSAP-2, as well as a third protein (“component C”) in the large precursor protein. J. A. Whitsett, personal communication.
for &Glucosidase
19599 REFERENCES
1. Ho, M. W., and O’Brien, J. S. (1971) Proc.Natl.Acad.Sci. U. S. A . 68,2810-2813 2. Basu, A., Glew, R. H., Daniels, L. B., and Clark, L. S. (1984) J . Biol. Chem. 259,1714-1719 3. Fujibayashi, S., Kao, F., Jones, C., Morse, H., Law,M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37,741-748 4. Berent, S. L., and Radin, N. S. (1981) Arch. Biochem. Biophys. 208,248-260 5. Iyer, S.S., Berent, S. L., and Radin, N. S. (1983) Biochim. Biophys. Acta 748, 1-7 6. Ranieri, E., Paton, B., and Poulos, A. (1986) Biochem. J. 2 3 3 , 763-772 7. Christomanou, H., Aignesberger,A., and Linke, R. P. (1986) Biol. Chem. Hoppe-Seyler 367,879-890 J., Glew, R. H.,Kuhlenschmidt, 8. Peters, S. P., Coyle, P., Coffee, C. M. S., Rosenfeld, L., and Lee, Y. C. (1977) J. Biol. Chem. 2 5 2 , 563-573 9. Wenger, D. A., and Roth, S. (1982) Biochem. Int. 5,705-710 10. Datta, S. C., and Radin, N. S. (1986) Lipids 2 1 , 702-709 11. Berent, S. L., and Radin, N. S. (1981) Biochirn. Biophys. Acta 664,572-582 12. Kleinschmidt, T., Christomanou, H., and Braunitzer, G. (1987) Biol. Chem. Hoppe-Seyler 368,1571-1578 13. Datta, S. C., and Radin, N. S. (1984) Anal. Biochem. 1 4 2 , 196203 14. Radin, N. S., and Berent, S. L. (1982) Methods Enzymol. 8 3 , 596-603 15. Redinbaugh, M. G., and Turley, R. B. (1986) Anal. Biochem. 1 6 3 , 267-271 16. Tarr, G. E. (1986) in Methods of Protein Microcharacterization (Shively, J. E., ed) pp. 155-194, Humana Press, Clifton, NJ 17. Sakamoto, Y., Kitamura, K., Yoshimura, K., Nishijima, T., and Uyemura, K. (1987) J. Biol. Chem. 262,4208-4214 18. Sano, A., and Radin, N. S. (1988) Biochem. J. 2 6 4 , 297-300 19. Sano, A., and Radin, N. S. (1988) Biochem. Biophys. Res. Commun. 154,1197-1203 20. Schroeder, W. A., Shelton, J. B., and Shelton, J. R. (1969) Arch. Biochem. Biophys. 130,551-556 21. Christomanou, H., Kleinschmidt, T., and Braunitzer, G . (1987) Biol. Chem. Hoppe-Seyler 368,1193-1196 22. Bause, E. (1983) Biochem. J. 2 0 9 , 331-336 23. Tarentino, A.L., and Plummer, T. H., Jr. (1987) Methods Enzymol. 1 3 8 , 770-778 24. Kite, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 6 7 , 105-132 25. Dewji, N. N., Wenger, D. A., and O’Brien, J. S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,8652-8656 26. Inui, K., Kao, F.-T., Fujibayashi, S., Jones, C., Morse, H. G., Law, M. L., and Wenger, D.A. (1985) Hum. Genet. 6 9 , 197200 27. Fujibayashi, S., Kao, F.-T., Jones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 3 7 , 741-748 28. Li, S.-C., Hirabayashi, Y., and Li, Y.-T. (1981) J. Biol. Chem. 256,6234-6240 29. O’Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241,1098-1101 30. Ho, M.W. (1974) in EnzymeTherapyin Lysosomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W. Th., eds) pp. 239-246, North-Holland Publishing Co., Amsterdam 31. Collard, M. W., and Griswold,M. D. (1987) Biochemistry 26, 3297-3303 32. Collard, M. W., Sylvester, S. R., Tsuruta, J. K., and Griswold, M. D. (1988) Biochemistry 27,4557-4564 33. Glasser, S. W., Korfhagen, T. R., Weaver, T., Pilot-Matias, T., Fox, J. L., and Whitsett, J. A. (1987) Proc. Natl. Acad. Sci. U. S. A . 84, 4007-4011 34. Furst, W., Machleidt, W., and Sandhoff, K. (1988) Biol. Chern. Hoppe-Seyler 369,317-328
19600
Activator Protein for 0-Glucosidase SUPPLEMENTARY MATERUU. TO THE ACTNATOR PROTEIN FOR GLUCOSYLCERAMIDE P-GLUCOSIDASE FROM GUINEA PIG LIVER IMPROVED ISOLATlON METHOD AND COMPLETE AMINO ACID SEQUENCE AKIRA SANO. NORMAN S.W I N . UNDA L. JOHNSON. AND GEORGE E. TARR
EXPERIMENTALPROCEDURES
Maferl~fs-HaRley gulnca plgs were decapltated end the llvers were frozen rapidly frozen guinea pig from Pel-Freez Biologicals (Rogers. A Z ) were also used. Wvcr Instead of spleen was chosen as the SAP-2 source because the concentratlon appears to be higher there (13) and because the connective tlssue In the spleens of large anlmals makes them dlfnrult to homogeme. Gulnea plg. rather than a large animal. was used as the Hver source because of the relatlvely short Ume needed to exclse the liver. Thls reduces the danger of partmonem changes. DEABSephacel and Sephadex G-75 Ifinel were from Pharmacla: erystalllne BS.4 Grade A. was from Calblochem: acetonltrile. HPLC grade. was from Flsher Sclenufic: TFA. HPLC/Spectro grade was from Fierce Chemical: triethylamlnc 9996 pure was from Aldrlch Chemical: peptlde N-glycosldase was from Boehrlnger Mannhelm: other matenals were from Slgma Chemical. SA€-2 A s s a m p l e s were assayed far Sumulatory actlvlty toward a paruauy punned preparatlon of glucosidase by lncubatlon with methylumbelllferyl glucoslde in acetate buffer. pH 4.5.with Trlton X-104as previously descrlbed 1141. Rotem Defemination and Amno Acld Analysi-The bicinchaninlc acld method 1151 was used for proteln measurement with BSA as standard. Ammo acid eamposltlond analysls was done with phenyllsathlocyanate derlvatizatlon by the methods of Tarr 116). Isolafion ofSAP-2"AU steps were camed out at rwm temperature except wheze Indlcated. In the run described here. thvty frozen llvers I378 9, were crushed in alumlnum foll with a wooden mallet and heated In a b u t 5 "01 of bolllng water far IO mln. Thls was dane to rmnlmlzc the danger of artifactual enzymatic degradation of SAP-2. The mtxture was homogenlred with an Ultra-Turrax unit (overhead motor. blades at bottom) whtle stlll hot. then the homogenate was cooled In ice cold water to rmm temperature and centrifuged 20 mln at 5000 x g. The resultant pellet was rehamogenlzed with 1512 ml of water and the supernatant portlon. after cenmfugatlon as above. was pwled with the &st extract. Ammonlum sulfate was added slowly. Wth sumng. to produce 45% Saturation and. alter srandlng an addluonal hour. the minure was eenuifuged 30 mm as above. Thls step was repeated with the supernatant portlon and 80% saturated iunmonlum sulfate and the resultant pellet was dlssdved In 60 ml of Buffer A.I and dialyzed against the same buffer. an dry Ice. then stored at -8OOC before use. Commerclally avallablc IlYerS
The sample was pumped at 33.6 d / h Into a column of Dm-Sephacel (2.6 X 37 cml previously equlllbrated with Buffer A and followed by 75 ml of Buffer A and 1200ml of the same buffer contdnlng a Unear gradlent of S0.7 M NaCl. A yellowish brown matenal eluted before SAP-2. with averlapplng. Fractions nch ln glucasldase-sumulatlnp actlvlty. eluung at -0.4 M NaCI. were paolcd and adjusted to 8046 saturatlon with ammonfum sulfate. After 1 h the suspension was centrifuged I5 mln at 1O.ooO x g and the pellet was dissolved in 8.2 ml of Buffer A conmnlng 0.1 M NaCl T h e sample was now pumped at 16 ml/h through a Sephadex G-75 column 12.6 x 34 cml. packed and eluted with the same buffer. Thls step [figure not shorn1 produced a relatlvely broad peak for SAP-2. a feature noted before with human and bovlne SAP-%. lndlcauve of heterogenelty In molecular welght. T h e SAP-2 fractlons. eluung between 91 and 128 ml. were pooled and concentrated by ammonlum sulfate preclpltatlon as before. The pellet was dlssolved In 1.5 ml of Buffer A/O.I M NaCI. then pumped at 10 mllh Into a column of Cad-Sepharose (1.3 x 4.5 cml previously equubrated with the same buffer and followed by 28 ml of the buffer and 60 ml of the buffer conlalnlng a linear gradlent of &1W mM methyl mannoslde. Three peaks with SAP-2 actmty were seen. the first two passlng through the column In the startlng buffer and the third eluting with dilute methyl mannoslde Wig. 1). Uslng a shorter ConA column. we had previously found only two peaks with human SAP-2 151.The SAP-2 fractlons whlch eluted after the gradient started were pooled. dialyzed agaihst water. and lyophlllzed.
e0
E
180
s .-
-z2
0
m
r?
0 180
m
1::
140
5
c
e
Ha
120
a 1000
5
10 15 20 2 5 3 0 3 5 Fraction Number
0
FIG. 1. C h m m t o g n p h y dth c011A~sFph.rosc.T h e fractlons 12.5m l l were assayed for absorbance ILmc AI and SAP-?actlvity (une BI. The horkontal bar shows the fractlons p l e d for reverse phase HPLC
T h e dry SAP-2 sample w a ~ now dlssalved In 0.1% TFA 1 - 5 mg/ml] and lnJected Into a 0 2 ml Imp In am HPLC system. Separatlan was accomplished with a ugh-paroslty Cg reverse phase column W t e c h Macrosphere 300 C4, 4.6 x 1 5 0 5 pm particlcsl.
mm.
monitored at 220 nm. The clutlon solvent was a h e a r gradlent of 55-656 acetonltrile in 0.1% TFA ThLs s t e p produced several overlapplng peaks of acuve material which almost colnelded with the W absorbance curve 21. T h e last part of the mam peak
Wig.
was used for further analyses. FraCtlonS were evaporated to dryness under vacuum In a cenuifuge ISpeedVac, Savant Instruments. Hlcksvllle. Nu) and dlssolved In 0.05% methylamine for activation assay. Tnethylarmne has been used earller for handlIng a hydrophoblc proteln (171. The m a h ~ C U V Cpeak fractlons were pooled In a polypropylene cenmiuge tube and lyophllbed for other analyses. PepUde fragments were purified slmllarly. The SAP-2 actlvlty was stable during C4 chromatography and dqing steps desplte the low pH and hlgh concentration of organlc solvent.
.
of ~ A P - T S
FIG. 2 . BPU:-no=
dth c, m e n c p h u c C O I ~ Wne . A shows the absorbance at 220 nm. The fractions ( 1 mll were assaved for enzyme actlvatlon nine 81
0
II
IO
Y
Time (mm)
Emluatlon of the Fw@c&n
Steps-As noted
Wth
prcvious pucUlcatlon methods.
SAP-2 specific actlvlty could not be d e t e d n e d with the enzyme stlmulatlon assay In the LNtlal exvact because of the presence of lnhlbltory material. even after ammonlum sulfate fractlonatlon.Approamate assays lndlcated that more than 85% of the SAP-2 preclpltated between 45 and 80% of saturatlon with ammonllum sulfate. The Lnhlbltory matenal eluted from the &on exchange column Just after the SAP-2. suggesting It Is a more acldlc substance than SAP-2. Recent work has shown that much of the Ulhibltlon 1s due to the presence of ribonucleic acld In the extract 1181.
The SAP-2 pumncd to the bal stage [Table I) had a specific actmty of 35 unIts/kg. slmllar to the one reported for bavlne spleen SAP-2 but Somewhat lower than that of human spleen SAP-2 151. TABLE I
mriricatim or sohydro1.s.
u n i t . / pngg
guinea p i g
Specific
Total Total prot.in
ire-
activity
.ctivity
1xv.r
Told
1378
g)
Yield
puririo.t1on
unit.
9
Crud. axtract 4930 -onium sulfat.877
DtAE-S.phac.1
75.4
1.0
100
176,000 12.0 11.7
3.7
12
A-S.phaT0.a
21.0 61,900 2.28
1.3
25
C4 Mltrosph.=.
0.69
S.phada= Con
0-76
245.000
21,100
3.25
34.9
10.7
9.8
SDS-PAGE of our preparatlon revealed three bands of 6.5. 8.5.and 10 kDa. vislble with sllver smnlng 1191. In our preuious study of the SAP~2from human spleen 151. the Cod-blndlng matellal yielded only two bands. with natlve PACE. The concentnuon of SAP-2 in guinea plg llver can he estlmated from the data ln Table I as being > I 8 pglg. This d u e Includes the acttvator protetn that does not bInd to concanavalin A. Previously publlshed concentratlons for total SAP-2 Isolated from normal human and bovine spleen. corrected for lossts, are slmllar 14.5).However the reported value for human hram. 14 wig. corrected for yield to 236 wglg 191. 15 much hlgher. Concentrations ln mouse Ilvcr. measured by enzyme-llnked Immunoassay 1131. were 3 . 6 4 . 6 pg/g. The maJor n m l features of our lsolatlon method are the use of procedures to mlnlmlze postmortem change. the use of ammonium sulfate as an lnltlal concentration and pu&flcatlon step. and the use of an HPLC reverse phase column. Deglymsyla(b~Th1swas done as described separately 1191. with protein N-glycosldasc F ~nEDTA and ephenanthrollne. Incubated for 10 h. The lncubatlon mkture was pumed with the C I column to remove the enzyme and salts. All of the W-absorblng material cluung after the INU& enzymefbuffer peak was utillred for sequenclng. The product showed a slnglc band of 4 kDa with SDSPAGE 1191. lndlcatlng suhstanUally complete rcmoval of the ollgosaccharldes from atl three forms of the activator to form a relatlvely homogeneous protein. Redwtloon and AUcylatlon-SAP-2 was reduced with dlthlothrcltal ~nguanidlne.HC1 and alkylated with vlnylpyridlnc or lodoacetlc acld 116). The reduced. allcylated SAP-2 was separated from the reagents with the C, column. the solvent wad removed by cenwfugal vacuum CvaporaUon and the resldues were dlssolved ~n 0.05% triethylamine. pooled. and lyophlllzed. CleoMethals"C1eavage of SAP-2 with CNBr was performed I n 70% fomie add (16) or 70% TFA (201 for 18 h at room temperature In the dark. The mactlon mlnure was lyophlllzcd. the resldue was dissolved In 0.1% TFA and the products were separated by HPLC as above. Dlgestlon of 580 pg of reduced. alkylated SAP-2 with 5 pg of trypam was canted O u t tn 0.1 ml of 0.1 M mmonlum blearbonate. pH 7.9. at 37'c for 2.5h. m e dlgcsted sample was acldffled to pH -2 with TFA and then subJected to HPLC CNBr fragmentatlon of 140 118 of the large peptlde denved from trypsln dlgesuon ( ' T p ) was performed In70%TFA. after whlch the solution was lyophllbed and the resldue fracuonated by HPLC as above. The large pepude from thla step was funhcr dlgested with staphylacoecal V8 protease 10.5 pgl In 0.1 ml of 50 mM ernmonlurn acetate pH 4.0. at 37'C for 18 h. The dlgcst was aeldllled with TFA and a p p k d to HPLC
m.w. Peptldc Tp I140 ggl was also dlgested with 5 pg of trypsln-free chpotrypsln In 0.1 ml of 0.1 M ammonlum blcarbanate pH 7.9. at 37'C for 7 min. The samplc WBS then acldlRed with F A and subJected to HPLC (Fig. 3BI.
19601
Activator Proteinfor P-Glucosidase
T i m (mnl
sequence DefematlOn-Automatic Edman degradatlon was carrled out In a model 47oA gas phase sequence analyzer and IZOA FTH analyzer lAppUed B l ~ ~ y s t e mManual ~l. sequence analyses wcrc performed using the p m t l o n method or the n h method with
Polybrene 1161.
The data show the number of residues in each peptide fragment, analytically determined and l i n parentheses] calculated from sequencing da:a.The four peptides / n the right 51de of the table were all derived from t h e l a r c , ~ trypsin-derived peptlde shown in t h e T p column. aThe CNBr produced hSer from M e t , vhrch overlapped G l y in the chromatograr b'rhls peptide was contaminated with a peptide beglnnrng vlth Sez-K-.
IUSULTS ~ r n t "A& ~
first few cycles of N-terrmnal analysls showed that
Sequen-rnc
The armno acld camposluonal analysls of SAP-2 (Table n11 agrees well with lhe
SAP-2 consisted of three polypeptides that dlffered by the absence or presence of the
nrst and second armno adds. m e malor peptlde possessed the sequence Glu-Ser-
results of sequencing. lndlcatlng that the sequence Is complete. A low content of
v a l - ~ h r - c y s .another pepllde began with Ser-Val-Thr-Cys. and the thlrd began with
aromatic armno a d d s was noted: Trp and Phe were absent. The absence of Trp was
Val-Thr-Cys. m e same sequence and mlxture were observed when mored forms of
establlshed fluarimemcally as well as by sequencing.
SAP-2 =re analyzed (when the Cod-Sepharose step wa8 omlttedl. Human A l a activator from Gaucher spleen was also found to cantam a
TABLE 111
sunllar mlxture of Amino
shortened forms (211.
acid composition of pin.. analytical data and
pig 1iv.C S M - 2 c.lcu1at.d from s.pu.ns. data.
from
Sequencing of gulnea plg llver SAP-2 revealed the presence of 81 amlno adds.
maklng the calculated Mr 8743. The pepudes used to determine the sequence are Indicated In
m. 4. Except for posluon 80. there p p u d e s overlap by at least three
4.1
rcsldues. IdenUAcaUons of all reslducs were conflrmcd by composlUonal armno acld
I*Im.m?,ID.
1,
""PI".,
1
IO I5 21 21 . I ".C.IC.,"I...*.L*D".",.,~~~".~~,"~.~~,,
,I
I,
40
..
I , -.-I I , -.-XI 0 ,-,a
A*"
2
I . " Iy. Y . t
Asp
4
Ph.
6
Pro
2
s . r Thr Trp Tyr V . 1
Ala
4
1.2
AT9
analysls (hble U] and dupllcatc sequence analysls of the peptides. 1
Analytical E.."l."
Analytical Calculated .."l.V C.."l.V
..................................................
............................
4.5
112 cy. Gln
GI" G1Y
110.7)b 3.1
Bi. 11.
1
11
0.a
3 1
3.1
4
CLlCu1at.d V.1Y.S
9.9 4.0
10
2.5 0.1 2.3 9.3
3 0
3.4
3
0.0 2.0
0
9.2
4
2
a
2 11
a Mole cqulvalents. assuming My = 8743.
b Sum of free a d d and amldated acld. C Average of three values. me
SIX Qs
residues were s h o w to be in disulfide llnkages. AlkylaUon of naUve and
reduced SAP-2 with vlnylpyndine. fallowed by amlno add analysls. showed the presence of s k pyidylcthyl-Cys resldues In the latter and none In the former. An attempt at alkylating natlve SAP-2 did not change the elutlon from the CI column. but FIG. 4. Sumrmrg of dm- for sequenced peptldu. m e dashes representldentlned sequences: the dots represent the remamlng ammo aclds ln the Isolated peptlde. All pepudes s h o w . except those of llne 9.wem formed from SAP-2 whlch had been deglycosylated. reduced with dllhlothreltol. and alkylated with vinylpycldlne or Iodoaceuc add. b e 1 Is derived fmm thls pmduct by gas phase sequenclng. UneS 2 and 9 are derived from cyanogen bromlde cleavage of the proteln. Une i Is from trypsin cleavage of the protern. Une p Is from one of the pepudes formed by CleaMge of the peptlde of Unc 4 by cyanogen bromlde. Unc fi Is from one of the other peptldes formed along wlth the peptlde above. cleaved by staphyloccal V8 protease. UneS 2 and k w e r e derived by ehymoUypsln dlgesuon of the tryptic pcptlde of Llne 4. Llne 9 was derived from CNEr reacuon in 7096 TTA with Intact SAP-2.
alkylation d e r reductlon resulted ln conversion to an earllcr-elutlng peak.
Study of the sugars In SAP-2 showed that It contans an N~llnked ollgosacchaide cham 1191 T h e cham appears to be attached to Asn-22 slnce the sequence Asn-kgThr IS the only trtpepude In S A P 2 which nts the Asn-X-mrlSer pattern common to all N-glycosldlcally llnked sequences [XIs any ammo add except Pro) 1221. Thls
observatlon Is also ln accord with our flndlng that sequenclng SAP-2 produced & a t pasltlon 22 after deglycosylauan with N~glycosldaseF. Thls occurs because the enzyme cleaves the amide Unkage (231. Moreover N-termlnal sequenclng of natlve
SAP-2. after cleavage with CNBr. showed a blank at posltlon 22. evidently hecause
glycosylated Asn Is normally lost In the procedure. SequcneIng the C-tcrrmnal reglon of SAP-2 requlred hvo approaches. Dlgestlan of peptlde Tp lllne 4. Flg. 4) with chymotrypsin yleldcd many peptldes (Flg. 381. severai
of wNeh were ldcnllfled by composltlonal analysls as belng part of the C-tcrmlnal reglon. However the sequenclng data of these pepudes were rellablc only up to
Ser-IK). Dlgestlon of Intact. unreduced SAP-2 with CNBr In 70% F A ylclded four peptldes were were sequenced without separation. ITFA was used because of Its superior cleavage of Met-Serllhr llnkages (20)).l b c N-terrmnal sequence and the two expected sequences following Met-16 and Met-66 were madlly Idenllflcd. In
addltlon. the C-termlnal dlpepude. Ser-Gly. could be unlquely ldentlned from thls M u r e . lhese data yleld a calculated content of three Gly resldues. In good
agreement with the analytlcal data for Intact SAP-2 mable 1111.
