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Properdin is a trimer
Molecular Immunology. Vol. 19, No. 4. pp. 631 to 635, 1982. Printed in Great Bntain.
0161.58YO,Q040631-0580300~0 0 1982 Pergamon Press Ltd.
PROPERDIN RICHARD MRC Immunochemistry South (First receiced
IS A TRIMER G. DISCIPIO*
Unit, Department of Biochemistry, Oxford Parks Road, Oxford OX1 3QU. U.K.
16 June 1981; accepted
in revisrdjbrm
12 Octohrr
University,
1981)
Abstract-Physical measurements on native properdin suggest that this protein is a trimer 145,6OC~152,600 consisting of three apparently identical subunits of mol. wt 49.100 each.
INTRODUCTION
Properdin is a basic plasma protein that is involved in stabilizing the labile C3 convertase of the alternative pathway of complement (Fearon & Austen, 1975; Medicus et al., 1976). Properdin has been reported to exist in two forms referred to as native and active. The native form can be converted into the active form without alteration in the primary structure by such means as freezing and thawing a properdin solution at -80°C (Medicus et al., 1976, 1980; Gijtze et al., 1977). Properdin was calculated originally to have a mol. wt of 220,000 (Pensky et al., 1968). Subsequently, Minta & Lepow (1974) reported the mol. wt to be 184,000, and they concluded that properdin was a tetramer of four apparently identical subunits of mol. wt 45,000 each. The subunit sizes as determined .from SDS-polyacrylamide gel electrophoresis experiments were reported to be 46,000 (Minta & Lzpow, 1974), 50,000 (Gatze et al., 1977) and 56,000 (Medicus et al., 1980). A reinvestigation of the quaternary structure of properdin was considered of value.
MATERIALS
AND
METHODS
Sephacryl S300 and Sepharose CL6B were products of Pharmacia Fine Chemicals Ltd, Hounslow, U.K. Guanidine HCl ultrapure was obtained from BRL, Rockville, Maryland. Morpholino-propanesulfonic acid (Mops)? and diethylpyrocarbonate were obtained from *Address correspondence to: R. G. DiScipio, Scripps Clinic and Research Institute, Department of Molecular Immunology, 10666 North Torrey Pines Road, La Jolla, CA 92031, U.S.A. 7 Abbreviation: Mops, morpholino-propanesulfonic acid.
of mol. wt
Sigma Chemical Co. Ltd, Poole, U.K. Dimethylsuberimidate and dimethyladipimidate were purchased from Pierce & Warriner Chemical Co. Ltd, Cheshire, U.K. Native properdin was purified by the method of DiScipio (1981). The purification steps included 5% polyethylene glycol precipitation of BaCl,-treated human plasma, CMSephadex C50 column chromatography, passage through DEAE-Sephadex A50, and passage through a column of rabbit anti-human IgG sepharose. The partial specific volume of properdin in non-denaturing buffer was calculated from the amino acid composition (Cohn & Edsall, 1943). The partial sp. vol of properdin dissolved in 6 M guanidine HCl solutions was calculated by the method of Lee & Timasheff (1974). The values of the partial sp. vols were corrected for the fraction of carbohydrate (Gibbons, 1966). The amino acid analyses and carbohydrate content of properdin have been previously reported (Minta & Lepow, 1974; Reid & Gagnon, 1981). The diffusion coefficient of properdin was determined by interpolation from a standard plot of the reciprocal of the diffusion coefficient (l/D) against the cubic root of the distribution coefficient (Kd)+ for marker proteins fractionated on Sephacryl S300 (2.6 x 95 cm) or Sepharose CL6B (2.6 x 95 cm). The columns were run in 10mM imidazole HCl buffer, pH 7.3, 0.35 M NaCl. The calibration standards were as follows: IgM, D20,w = 1.7 x lo-’ cm2/ set; thyroglobin, D20,w = 2.5 x lo-’ cm2/sec; IgG, apoferritin, D,,,, = 3.6 x lo-‘cm’/sec; catalase, D20,w D 20,w = 4.0 x lo-’ cm2/sec; = 4.1 x lo-’ cm2/sec; bovine serum albumin, = 5.9 x lo-’ cm2/sec; ovalbumin, DzO w D I”i.i x lo-’ cm2/sec. The value for the diffusion coefficient of IgM was taken from Beale 631
RICHARD
632
G. DISCIPIO
& Buttress (1969). The diflusion coefficients of the other standard proteins were taken From Andrews (1965). Fractions eluted from the column were monitored for properdin haemolytic activity. calibrated gel filtration columns have been demonstrated to provide accurate determinations of Stokes radii and diffusion coefficients (Ackers. 1964, 1975; Andrews, 1965). The sedimentation coefficient of properdin was determined by employing a Beckman Model E analytical ultracentrifuge that was equipped with an electronic speed control and a photoeIe~tric scanner. A properdin sample of 0.2 mg/ml in 10 mhil Mops--Tris buffer, pH 7.3. 0.15 M NaCl was centrifuged at 52,000 rpm at 20°C in a 12 mm double-sector cell. Absorption optics were used to monitor the rate of sedimentation, The sedimentation value attained was corrected to standard conditions to obtain szo, W. The mol. wt (Mf of properdin was calculated from szO,w and DZO,w measurements by the Svedberg equation: &f-__-_-_.
R T.s D(l - up)
The mol. wt of properdin was also determined by meniscus depletion sedimentation ultracentrifugation (Yphantis, equilibrium 1964). The sedimentation equilibrium experiment was performed by using a six-channel Kel-F centerpiece. The properdin samples were monitored at three different concentrations (0.2, 0.15 and 0.1 mg/ml) by use of absorption optics. The buffer was 10mM Mops-Tris, pH 7.3.0.15 M NaCl and the samples were centrifuged at 15,000 rpm at 19.4”C for 36 hr. The protein concentration (c) was expressed in units of absorption at 280nm, and the mol. wt was calculated from the equation: 2RT_-..__ d_._In c M = ~_ (1 - Gp)ro2 dr2 ’ The mol. wt of the subunits of properdin was determined by dissolving salt-free lyophilized properdin in 20 mM sodium phosphate buffer, pH 7, 6 ,V guanidine HCl. The meniscus depletion ultracentrifugation method as described earlier was used except that the rotor speed was 22,00Orpm, and the temperature was 21.2”C. SDS-polyacrylamide gel electrophoresis was performed by the method of Weber & Osborn
(1969) as modified by Kisiel rt al. (1976). The subunit mol. wt of properdin was estimated from semilog plots of apparent mol. wt against distance of migration using the following protein standards: phosphorylase a, 97,O~; bovine serum albumin, 68,000; ovalbumin, 45,000; bovine carbonic anhydrase. 29,000; myoglobin, 17.000. The frictional ratio was determined as described by Ackers (1975). The extinction coefficient was determined by employing a Beckman Model E analytical ultracentrifuge to determine protein concentrat~on by synthetic bouildary experiments (Babul & Stellwagen, 1969). The protein samples were in 10 mM Mops-Tris buffer, pH 7.3, 0.15 M NaCl. The CID at 280 nm was corrected for Rayleigh light scattering as described by Shapiro & Waugh (1966). Chemical ~rosslinking studies were attempted by incubating properdin (0.3 mgjml) in 0.1 hii sodium phosphate buffer, pH 8, with various concentrations of diethylpyrocarbonate, dimethylsuberimidate or dimethyladipimidate up to a final concentration of 50 mM. The reactions were allowed to proceed for 3 hr at 37°C and the samples were dialysed against several volumes of 0.1 M sodium phosphate buffer before being subjected to SDS-polyacryIamide ge’ electrophoresls’ RESULTS
Sedimentation velocity ultra~entrifugation analysis provided a sedimentation coefficient of 5.0 S, which is similar to the results of Pensky et al. (1968) and Minta & Lepow (1974). There was no observed tendency toward aggregation. The diffusion coefficient of native properdin was determined by gel filtration. The standard plot of the reciprocal of the diffusion coefficient (l/O) against the cubic root of the distribution ~oe~cient (l
Properdin
633
is a Trimer
pyrocarbonate were unsuccessful. There was no detectable crosslinking with any of the reagents tested. DISCUSSION
I
I
I
I
I
I
04
05
06
07
08
09
lk,
lh
Fig. 1. The plot of the reciprocal of the diffusion coefficient (l/D) against the cubic root of the distribution coefficient (KJ:. The reciprocal of the diffusion coefficient of properdin (A) was estimated by gel filtration on Sephacryl S300 calibrated with the following standards (0) in the order of increasing K,: IgM. thyroglobulin apoferritin, IgG, catalase, bovine serum albumin and ovalbumin.
intact properdin was estimated is shown in Fig. 2. The results of three determinations indicated a mol. wt of 145,600 + 8100. Figure 3 shows a graph of In c against r2 for the determination of the mol. wt of the subunit size of properdin. The results of three determinations indicated a mol. wt of 49,100 k 3200. Table 1 provides a compilation of the various physical parameters of human properdin. It is concluded that properdin is a trimer. Efforts to crosslink properdin with dimethylsuberimidate, dimethyladipimidate and diethyl-
On the basis of sedimentation velocity ultraequilibrium centrifugation, sedimentation ultracentrifugation, and diffusion coefficient measurements that are reported in this paper, it is concluded that native properdin has a mol. wt of 145,6O(r152,600, and consists of three apparently identical subunits of mol. wt 49,100 each. The mol. wt of intact properdin is in disagreement with the work of others. Pensky et al. (1968) observed a diffusion coefficient, D 20,.,, of 2.17 x lo-’ cm2/sec for properdin. By employing this diffusion coefficient with a sedimentation coefficient, szO. w, of 5.2S, and an assumed value for the partial sp. vol of 0.75 ml/g, the mol. wt for intact properdin was calculated to be 223,000. The more accurate value for the partial sp. vol, based on the amino acid and carbohydrate content, is 0.700mljg. Employing the corrected value for the partial sp. vol along with the s20,W and values determined by Pensky et al. (9%: , a mol. wt cf 184,000 can be calculated. The determination of the sedimentation coefficient of native properdin reported here, s~,,~ = 5.OS, is in reasonable agreement with the value of s20,W = 5.2s determined by Pensky et al. (1968), but the diffusion coefficient, D,,, w, of 2.66 x lo-’ cm2/sec reported in this paper, based on gel filtration analysis, differs markedly from the value reported by Pensky et al. used boundary spreading of (1968), who 20-
IO-
OI u -10 c
/
-2o-
34 Fig. 2. The plot of In c against r2 for the sedimentation ultracentrifugation experiment for one sample of native properdin. The protein concentration was 0.2 mg/ml in 10 mM MopssTris, pH 7.3, 0.15 M NaCI. The slope of the graph gave the value of d(In c)/dr’ from which the mol. wt was calculated, Three samples of properdin were studied and the results were averaged.
I
I
345
35
I 35 5 2
I
I
I
36
365
37
I
375
Fig. 3. The plot of In c against r2 for the sedimentation equilibrium ultracentrifugation of one sample of denatured properdin. The properdin (0.2 mg/ml) was in 1OmM sodium phosphate buffer, pH 7.0, 6 A4 guanidine HCI. Three samples of properdin were studied and the results were averaged.
634
RICHARD
Table
1. The physical
G. DISCIPIO
parameters
for human
properdin
Partial specific volume
=
0.700 ml/gm
Partial specific volume (6M-guanidine)
=
0.675 ml/gm
Sedimentation coefficient
s20,w =
5.0 s
Diffusion coefficient
D 2. w =
2.66 x 1O-7 cm 2fsec
mol. wt.
=
152,600 daltons
Mol. Wt. from sedimentation equilibrium
mol. wt.
=
145,600 * 8,100 daltons
Mol. Wt. from sedimentation equilibrium (6M-guanidine)
mol
=
49,100 + 3,200 daltons
Mol. Wt. SDS-polyacrylamide gels
mol. wt.
=
56,000 daltons
Frictional ratio
f/f0
=
2.35
Extinction coefficient
1% *28Onm,lcm
q
Mol. Wt. based on S20 w and D20 w
,
Schlieren optical patterns from an analytical ultracentrifugation experiment to determine their value. However, as no experimental details were published relating to their diffusion coefficient measurement, it is not possible to account for the discrepancy in the determination of this parameter. Minta & Lepow (1974) reported a mol. wt of 184,000 + 12,000 for intact properdin from equilibrium measurements. sedimentation Using a similar technique, we observed a value of 145,600 f 8100. The reason for this difference is not known. Measurements of the subunit size of properdin based on SDS-polyacrylamide gel electrophoresis gave a value of 56,000 in confirmation of the results of Medicus et al. (1980). The subunit mol. wt as determined by sedimentation equilibrium ultracentrifugation provided a mol. wt of the properdin subunits of 49,100 k 3200. The value of 49,100 is considered to be more accurate than the mol. wt estimated by SDS-polyacrylamide gel electrophoresis because properdin is a glycoprotein, and glycoproteins are known to migrate aberrantly on SDS-polyacrylamide gels as a result of the fact that glycoproteins bind less SDS than apoproteins (Segrest & Jackson, 1972). Properdin has been reported to exist in two forms referred to as native properdin and active properdin. Native properdin does not induce the depletion of C3 in properdin-depleted serum. but active properdin has been reported to do so (Giitze et crl., 1977). Native properdin has been reported to absorb to Sepharose-C3b. and this native protein can be eluted with 0.4 M NaCl. Active properdin may bind to Sepharose-C3b. but it cannot be eluted
’
wt
’
17"
with 2.0 M NaCl or 2.0 M guanidine HCl (Medicus et al., 1980). The properdin that was used in this work was considered to be in the native form as incubation of 15 pg of this protein with 1.0 ml of properdin-depleted plasma with 3 mM MgClz for 30 min at 37°C did not cause the depletion of C3 from this plasma. Furthermore, the properdin used in this work can bind to SepharoseX3b in 20mM imidazole HCl buffer, pH 7.3, 0.075 M NaCl, and all the absorbed protein can be eluted in the same buffer with 0.4 M NaCl. It is unlikely that the discrepancies in the data published here with previous reports are a result of one research group evaluating parameters for native properdin and another investigating activated properdin because the sedimentation coefficient of properdin was found to be szo_, = 5.&5.2S by. all investigators, and no tendency toward aggregation was observed during sedimentation analysis. Furthermore, there is no alteration in the primary structure of properdin upon activation (Medicus et al., 1980). The subunits of properdin are believed to be identical as extensive primary sequence analysis has revealed only one type of polypeptide chain (Medicus et al., 1980; Reid & Gagnon, 1981). Although the three subunits are apparently identical, DiScipio (1981) observed that native properdin binds zymosan-bound C3b with a one-to-one stoichiometry. It is suggested that the subunits are arranged in a specific fashion to construct a single C3b binding site. The value for the frictional ratio flfo = 2.35 is very similar to that of fibrinogen. Although this parameter is a function of hydration and
Properdin
molecular asvmmetrv. the value is sufficientlv large to war&t the yonclusion that properdii exhibits a rod-shaped structure. The failure to crosslink the properdin subunits with any of the chemical methods described and the high frictional coefficient of this protein suggest that the three chains of properdin are not arranged as an assembly of globular subunits, but the chains probably are coiled around each other. As oronerdin has a high content of proline and glycine (Reid & Gagnon, 1981), it is plausible that some of the attributes of the properdin molecule will be similar to features exhibited by the collagen triple helix or the polyproline helix. Future structural work on this orotein would be of great value in elucidating the role of properdin in the complement system. Acknowledgements-The Heritage for excellent cal ultracentrifuge.
author would like to thank Jane technical assistance with the analyti-
REFERENCES Ackers G. K. (1964) Molecular exclusion and restricted diffusion processes in molecular sieve chromatography. Biochemistry 3, 723~730. Ackers G. K. (1975) Molecular sieve methods of analysis. In The Proteins (Edited by Neurath H., Hill R. L. & Boeder C. L.), Vol. 1, p. 1. Academic Press, New York. Andrews P. (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606. Babul J. & Stellwagen E. (1969) Measurements of protein concentration with interference optics. Analyt. Biochem. 28,21&221. Beale D. & Buttress N. (1969) Studies on a human 19-s immunoglobulin M. Biochim. biophys. Acta 181, 25&267. Cohn E. J. & Edsall J. T. (1943) Density and apparent specific volume of proteins. In Proteins, Amino Acids & Peptides (Edited by Cohn E. J. & Edsall J. T.), p. 372. Reinhold, New York.
is a Trimer
635
DiScipio R. G. (1981) The binding of complement proteins C5,’ factor B, BlH, and prop&din to- C3b bound to zymcsan. Biochem. J. 199,485-496. Fearon D. T. & Austen K. F. (1975) Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J. exp. Med. 142, 856863. Gibbons R. A. (1966) Physico-chemical methods for the determination of the purity, molecular size and shape of glycoproteins. In Glycoproreins (Edited by Gottschalk A.), Vol. 5, p. 78. Elsevier Press, New York. GGtze O., Medicus R. G. & Miiller-Eberhard H. J. (1977) Alternative pathway of complement: nonenzymatic reversible transition of precursor properdin to active properdin. J. Immun. 118; 525-528. Kisiel W., Ericsson L. H. & Davie E. W. (1976) Proteolytic activation of protein C from bovine plasma. BiochemisWJ 15, 48934900. Lee J. C. & Timasheff S. N. (1974) The calculation of partial specific volumes of proteins in guanidine hydrochloride. Archs Biochem. Biophys. 165, 268-273. Medicus R. G.. Esser A. F.. Fernandez H. N. & MiillerEberhard H. J. (1980) Native and activated properdin: interconvertibility and identity of amino and carboxyterminal sequences. J. Immun. 124. 602-606. Medicus R. e., GGtze 0. & Miller-Eberhard H. J. (1976) Alternative pathway of complement: recruitment of precursor properdin by the labile C3jC5 convertase and the .potentiation of the pathway. J. exp. Med. 144, 1076-1093. Minta J. 0. & Lepow I. H. (1974) Studies on the subunit structure of human properdin. Immunochemistry 11, 361-368. Pensky J., Hinz C. F., Todd E. W., Wedgewood R. T., Boyer J. T. & Lepow I. H. (1968) Properties of highly purified human properdin. J. Immun. 100, 142-158. Reid K. B. M. & Gaanon J. (1981) Amino acid sequence studies of human properdin: N-terminal sequence gnalysis and alignment of the fragments produced by limited proteolysis with trypsin and the peptides produced by cyanogen bromide treatment. Molec. Immun. 18, 949-959. Segrest J. P. & Jackson R. L. (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Meth. Enzym. 28, 54-63. Shaoiro S. S. & Waueh D. F. (1966) The purification of himan prothrombL. Thromb. Diath. haemorrh. 16, 469-490. Weber K. & Osborn M. (1969) The reliability of molecular weight determinations by sodium dodecyl sulfate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. Yphantis D. A. (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3, 297-3 17.
0161.58YO,Q040631-0580300~0 0 1982 Pergamon Press Ltd.
PROPERDIN RICHARD MRC Immunochemistry South (First receiced
IS A TRIMER G. DISCIPIO*
Unit, Department of Biochemistry, Oxford Parks Road, Oxford OX1 3QU. U.K.
16 June 1981; accepted
in revisrdjbrm
12 Octohrr
University,
1981)
Abstract-Physical measurements on native properdin suggest that this protein is a trimer 145,6OC~152,600 consisting of three apparently identical subunits of mol. wt 49.100 each.
INTRODUCTION
Properdin is a basic plasma protein that is involved in stabilizing the labile C3 convertase of the alternative pathway of complement (Fearon & Austen, 1975; Medicus et al., 1976). Properdin has been reported to exist in two forms referred to as native and active. The native form can be converted into the active form without alteration in the primary structure by such means as freezing and thawing a properdin solution at -80°C (Medicus et al., 1976, 1980; Gijtze et al., 1977). Properdin was calculated originally to have a mol. wt of 220,000 (Pensky et al., 1968). Subsequently, Minta & Lepow (1974) reported the mol. wt to be 184,000, and they concluded that properdin was a tetramer of four apparently identical subunits of mol. wt 45,000 each. The subunit sizes as determined .from SDS-polyacrylamide gel electrophoresis experiments were reported to be 46,000 (Minta & Lzpow, 1974), 50,000 (Gatze et al., 1977) and 56,000 (Medicus et al., 1980). A reinvestigation of the quaternary structure of properdin was considered of value.
MATERIALS
AND
METHODS
Sephacryl S300 and Sepharose CL6B were products of Pharmacia Fine Chemicals Ltd, Hounslow, U.K. Guanidine HCl ultrapure was obtained from BRL, Rockville, Maryland. Morpholino-propanesulfonic acid (Mops)? and diethylpyrocarbonate were obtained from *Address correspondence to: R. G. DiScipio, Scripps Clinic and Research Institute, Department of Molecular Immunology, 10666 North Torrey Pines Road, La Jolla, CA 92031, U.S.A. 7 Abbreviation: Mops, morpholino-propanesulfonic acid.
of mol. wt
Sigma Chemical Co. Ltd, Poole, U.K. Dimethylsuberimidate and dimethyladipimidate were purchased from Pierce & Warriner Chemical Co. Ltd, Cheshire, U.K. Native properdin was purified by the method of DiScipio (1981). The purification steps included 5% polyethylene glycol precipitation of BaCl,-treated human plasma, CMSephadex C50 column chromatography, passage through DEAE-Sephadex A50, and passage through a column of rabbit anti-human IgG sepharose. The partial specific volume of properdin in non-denaturing buffer was calculated from the amino acid composition (Cohn & Edsall, 1943). The partial sp. vol of properdin dissolved in 6 M guanidine HCl solutions was calculated by the method of Lee & Timasheff (1974). The values of the partial sp. vols were corrected for the fraction of carbohydrate (Gibbons, 1966). The amino acid analyses and carbohydrate content of properdin have been previously reported (Minta & Lepow, 1974; Reid & Gagnon, 1981). The diffusion coefficient of properdin was determined by interpolation from a standard plot of the reciprocal of the diffusion coefficient (l/D) against the cubic root of the distribution coefficient (Kd)+ for marker proteins fractionated on Sephacryl S300 (2.6 x 95 cm) or Sepharose CL6B (2.6 x 95 cm). The columns were run in 10mM imidazole HCl buffer, pH 7.3, 0.35 M NaCl. The calibration standards were as follows: IgM, D20,w = 1.7 x lo-’ cm2/ set; thyroglobin, D20,w = 2.5 x lo-’ cm2/sec; IgG, apoferritin, D,,,, = 3.6 x lo-‘cm’/sec; catalase, D20,w D 20,w = 4.0 x lo-’ cm2/sec; = 4.1 x lo-’ cm2/sec; bovine serum albumin, = 5.9 x lo-’ cm2/sec; ovalbumin, DzO w D I”i.i x lo-’ cm2/sec. The value for the diffusion coefficient of IgM was taken from Beale 631
RICHARD
632
G. DISCIPIO
& Buttress (1969). The diflusion coefficients of the other standard proteins were taken From Andrews (1965). Fractions eluted from the column were monitored for properdin haemolytic activity. calibrated gel filtration columns have been demonstrated to provide accurate determinations of Stokes radii and diffusion coefficients (Ackers. 1964, 1975; Andrews, 1965). The sedimentation coefficient of properdin was determined by employing a Beckman Model E analytical ultracentrifuge that was equipped with an electronic speed control and a photoeIe~tric scanner. A properdin sample of 0.2 mg/ml in 10 mhil Mops--Tris buffer, pH 7.3. 0.15 M NaCl was centrifuged at 52,000 rpm at 20°C in a 12 mm double-sector cell. Absorption optics were used to monitor the rate of sedimentation, The sedimentation value attained was corrected to standard conditions to obtain szo, W. The mol. wt (Mf of properdin was calculated from szO,w and DZO,w measurements by the Svedberg equation: &f-__-_-_.
R T.s D(l - up)
The mol. wt of properdin was also determined by meniscus depletion sedimentation ultracentrifugation (Yphantis, equilibrium 1964). The sedimentation equilibrium experiment was performed by using a six-channel Kel-F centerpiece. The properdin samples were monitored at three different concentrations (0.2, 0.15 and 0.1 mg/ml) by use of absorption optics. The buffer was 10mM Mops-Tris, pH 7.3.0.15 M NaCl and the samples were centrifuged at 15,000 rpm at 19.4”C for 36 hr. The protein concentration (c) was expressed in units of absorption at 280nm, and the mol. wt was calculated from the equation: 2RT_-..__ d_._In c M = ~_ (1 - Gp)ro2 dr2 ’ The mol. wt of the subunits of properdin was determined by dissolving salt-free lyophilized properdin in 20 mM sodium phosphate buffer, pH 7, 6 ,V guanidine HCl. The meniscus depletion ultracentrifugation method as described earlier was used except that the rotor speed was 22,00Orpm, and the temperature was 21.2”C. SDS-polyacrylamide gel electrophoresis was performed by the method of Weber & Osborn
(1969) as modified by Kisiel rt al. (1976). The subunit mol. wt of properdin was estimated from semilog plots of apparent mol. wt against distance of migration using the following protein standards: phosphorylase a, 97,O~; bovine serum albumin, 68,000; ovalbumin, 45,000; bovine carbonic anhydrase. 29,000; myoglobin, 17.000. The frictional ratio was determined as described by Ackers (1975). The extinction coefficient was determined by employing a Beckman Model E analytical ultracentrifuge to determine protein concentrat~on by synthetic bouildary experiments (Babul & Stellwagen, 1969). The protein samples were in 10 mM Mops-Tris buffer, pH 7.3, 0.15 M NaCl. The CID at 280 nm was corrected for Rayleigh light scattering as described by Shapiro & Waugh (1966). Chemical ~rosslinking studies were attempted by incubating properdin (0.3 mgjml) in 0.1 hii sodium phosphate buffer, pH 8, with various concentrations of diethylpyrocarbonate, dimethylsuberimidate or dimethyladipimidate up to a final concentration of 50 mM. The reactions were allowed to proceed for 3 hr at 37°C and the samples were dialysed against several volumes of 0.1 M sodium phosphate buffer before being subjected to SDS-polyacryIamide ge’ electrophoresls’ RESULTS
Sedimentation velocity ultra~entrifugation analysis provided a sedimentation coefficient of 5.0 S, which is similar to the results of Pensky et al. (1968) and Minta & Lepow (1974). There was no observed tendency toward aggregation. The diffusion coefficient of native properdin was determined by gel filtration. The standard plot of the reciprocal of the diffusion coefficient (l/O) against the cubic root of the distribution ~oe~cient (l
Properdin
633
is a Trimer
pyrocarbonate were unsuccessful. There was no detectable crosslinking with any of the reagents tested. DISCUSSION
I
I
I
I
I
I
04
05
06
07
08
09
lk,
lh
Fig. 1. The plot of the reciprocal of the diffusion coefficient (l/D) against the cubic root of the distribution coefficient (KJ:. The reciprocal of the diffusion coefficient of properdin (A) was estimated by gel filtration on Sephacryl S300 calibrated with the following standards (0) in the order of increasing K,: IgM. thyroglobulin apoferritin, IgG, catalase, bovine serum albumin and ovalbumin.
intact properdin was estimated is shown in Fig. 2. The results of three determinations indicated a mol. wt of 145,600 + 8100. Figure 3 shows a graph of In c against r2 for the determination of the mol. wt of the subunit size of properdin. The results of three determinations indicated a mol. wt of 49,100 k 3200. Table 1 provides a compilation of the various physical parameters of human properdin. It is concluded that properdin is a trimer. Efforts to crosslink properdin with dimethylsuberimidate, dimethyladipimidate and diethyl-
On the basis of sedimentation velocity ultraequilibrium centrifugation, sedimentation ultracentrifugation, and diffusion coefficient measurements that are reported in this paper, it is concluded that native properdin has a mol. wt of 145,6O(r152,600, and consists of three apparently identical subunits of mol. wt 49,100 each. The mol. wt of intact properdin is in disagreement with the work of others. Pensky et al. (1968) observed a diffusion coefficient, D 20,.,, of 2.17 x lo-’ cm2/sec for properdin. By employing this diffusion coefficient with a sedimentation coefficient, szO. w, of 5.2S, and an assumed value for the partial sp. vol of 0.75 ml/g, the mol. wt for intact properdin was calculated to be 223,000. The more accurate value for the partial sp. vol, based on the amino acid and carbohydrate content, is 0.700mljg. Employing the corrected value for the partial sp. vol along with the s20,W and values determined by Pensky et al. (9%: , a mol. wt cf 184,000 can be calculated. The determination of the sedimentation coefficient of native properdin reported here, s~,,~ = 5.OS, is in reasonable agreement with the value of s20,W = 5.2s determined by Pensky et al. (1968), but the diffusion coefficient, D,,, w, of 2.66 x lo-’ cm2/sec reported in this paper, based on gel filtration analysis, differs markedly from the value reported by Pensky et al. used boundary spreading of (1968), who 20-
IO-
OI u -10 c
/
-2o-
34 Fig. 2. The plot of In c against r2 for the sedimentation ultracentrifugation experiment for one sample of native properdin. The protein concentration was 0.2 mg/ml in 10 mM MopssTris, pH 7.3, 0.15 M NaCI. The slope of the graph gave the value of d(In c)/dr’ from which the mol. wt was calculated, Three samples of properdin were studied and the results were averaged.
I
I
345
35
I 35 5 2
I
I
I
36
365
37
I
375
Fig. 3. The plot of In c against r2 for the sedimentation equilibrium ultracentrifugation of one sample of denatured properdin. The properdin (0.2 mg/ml) was in 1OmM sodium phosphate buffer, pH 7.0, 6 A4 guanidine HCI. Three samples of properdin were studied and the results were averaged.
634
RICHARD
Table
1. The physical
G. DISCIPIO
parameters
for human
properdin
Partial specific volume
=
0.700 ml/gm
Partial specific volume (6M-guanidine)
=
0.675 ml/gm
Sedimentation coefficient
s20,w =
5.0 s
Diffusion coefficient
D 2. w =
2.66 x 1O-7 cm 2fsec
mol. wt.
=
152,600 daltons
Mol. Wt. from sedimentation equilibrium
mol. wt.
=
145,600 * 8,100 daltons
Mol. Wt. from sedimentation equilibrium (6M-guanidine)
mol
=
49,100 + 3,200 daltons
Mol. Wt. SDS-polyacrylamide gels
mol. wt.
=
56,000 daltons
Frictional ratio
f/f0
=
2.35
Extinction coefficient
1% *28Onm,lcm
q
Mol. Wt. based on S20 w and D20 w
,
Schlieren optical patterns from an analytical ultracentrifugation experiment to determine their value. However, as no experimental details were published relating to their diffusion coefficient measurement, it is not possible to account for the discrepancy in the determination of this parameter. Minta & Lepow (1974) reported a mol. wt of 184,000 + 12,000 for intact properdin from equilibrium measurements. sedimentation Using a similar technique, we observed a value of 145,600 f 8100. The reason for this difference is not known. Measurements of the subunit size of properdin based on SDS-polyacrylamide gel electrophoresis gave a value of 56,000 in confirmation of the results of Medicus et al. (1980). The subunit mol. wt as determined by sedimentation equilibrium ultracentrifugation provided a mol. wt of the properdin subunits of 49,100 k 3200. The value of 49,100 is considered to be more accurate than the mol. wt estimated by SDS-polyacrylamide gel electrophoresis because properdin is a glycoprotein, and glycoproteins are known to migrate aberrantly on SDS-polyacrylamide gels as a result of the fact that glycoproteins bind less SDS than apoproteins (Segrest & Jackson, 1972). Properdin has been reported to exist in two forms referred to as native properdin and active properdin. Native properdin does not induce the depletion of C3 in properdin-depleted serum. but active properdin has been reported to do so (Giitze et crl., 1977). Native properdin has been reported to absorb to Sepharose-C3b. and this native protein can be eluted with 0.4 M NaCl. Active properdin may bind to Sepharose-C3b. but it cannot be eluted
’
wt
’
17"
with 2.0 M NaCl or 2.0 M guanidine HCl (Medicus et al., 1980). The properdin that was used in this work was considered to be in the native form as incubation of 15 pg of this protein with 1.0 ml of properdin-depleted plasma with 3 mM MgClz for 30 min at 37°C did not cause the depletion of C3 from this plasma. Furthermore, the properdin used in this work can bind to SepharoseX3b in 20mM imidazole HCl buffer, pH 7.3, 0.075 M NaCl, and all the absorbed protein can be eluted in the same buffer with 0.4 M NaCl. It is unlikely that the discrepancies in the data published here with previous reports are a result of one research group evaluating parameters for native properdin and another investigating activated properdin because the sedimentation coefficient of properdin was found to be szo_, = 5.&5.2S by. all investigators, and no tendency toward aggregation was observed during sedimentation analysis. Furthermore, there is no alteration in the primary structure of properdin upon activation (Medicus et al., 1980). The subunits of properdin are believed to be identical as extensive primary sequence analysis has revealed only one type of polypeptide chain (Medicus et al., 1980; Reid & Gagnon, 1981). Although the three subunits are apparently identical, DiScipio (1981) observed that native properdin binds zymosan-bound C3b with a one-to-one stoichiometry. It is suggested that the subunits are arranged in a specific fashion to construct a single C3b binding site. The value for the frictional ratio flfo = 2.35 is very similar to that of fibrinogen. Although this parameter is a function of hydration and
Properdin
molecular asvmmetrv. the value is sufficientlv large to war&t the yonclusion that properdii exhibits a rod-shaped structure. The failure to crosslink the properdin subunits with any of the chemical methods described and the high frictional coefficient of this protein suggest that the three chains of properdin are not arranged as an assembly of globular subunits, but the chains probably are coiled around each other. As oronerdin has a high content of proline and glycine (Reid & Gagnon, 1981), it is plausible that some of the attributes of the properdin molecule will be similar to features exhibited by the collagen triple helix or the polyproline helix. Future structural work on this orotein would be of great value in elucidating the role of properdin in the complement system. Acknowledgements-The Heritage for excellent cal ultracentrifuge.
author would like to thank Jane technical assistance with the analyti-
REFERENCES Ackers G. K. (1964) Molecular exclusion and restricted diffusion processes in molecular sieve chromatography. Biochemistry 3, 723~730. Ackers G. K. (1975) Molecular sieve methods of analysis. In The Proteins (Edited by Neurath H., Hill R. L. & Boeder C. L.), Vol. 1, p. 1. Academic Press, New York. Andrews P. (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595-606. Babul J. & Stellwagen E. (1969) Measurements of protein concentration with interference optics. Analyt. Biochem. 28,21&221. Beale D. & Buttress N. (1969) Studies on a human 19-s immunoglobulin M. Biochim. biophys. Acta 181, 25&267. Cohn E. J. & Edsall J. T. (1943) Density and apparent specific volume of proteins. In Proteins, Amino Acids & Peptides (Edited by Cohn E. J. & Edsall J. T.), p. 372. Reinhold, New York.
is a Trimer
635
DiScipio R. G. (1981) The binding of complement proteins C5,’ factor B, BlH, and prop&din to- C3b bound to zymcsan. Biochem. J. 199,485-496. Fearon D. T. & Austen K. F. (1975) Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase. J. exp. Med. 142, 856863. Gibbons R. A. (1966) Physico-chemical methods for the determination of the purity, molecular size and shape of glycoproteins. In Glycoproreins (Edited by Gottschalk A.), Vol. 5, p. 78. Elsevier Press, New York. GGtze O., Medicus R. G. & Miiller-Eberhard H. J. (1977) Alternative pathway of complement: nonenzymatic reversible transition of precursor properdin to active properdin. J. Immun. 118; 525-528. Kisiel W., Ericsson L. H. & Davie E. W. (1976) Proteolytic activation of protein C from bovine plasma. BiochemisWJ 15, 48934900. Lee J. C. & Timasheff S. N. (1974) The calculation of partial specific volumes of proteins in guanidine hydrochloride. Archs Biochem. Biophys. 165, 268-273. Medicus R. G.. Esser A. F.. Fernandez H. N. & MiillerEberhard H. J. (1980) Native and activated properdin: interconvertibility and identity of amino and carboxyterminal sequences. J. Immun. 124. 602-606. Medicus R. e., GGtze 0. & Miller-Eberhard H. J. (1976) Alternative pathway of complement: recruitment of precursor properdin by the labile C3jC5 convertase and the .potentiation of the pathway. J. exp. Med. 144, 1076-1093. Minta J. 0. & Lepow I. H. (1974) Studies on the subunit structure of human properdin. Immunochemistry 11, 361-368. Pensky J., Hinz C. F., Todd E. W., Wedgewood R. T., Boyer J. T. & Lepow I. H. (1968) Properties of highly purified human properdin. J. Immun. 100, 142-158. Reid K. B. M. & Gaanon J. (1981) Amino acid sequence studies of human properdin: N-terminal sequence gnalysis and alignment of the fragments produced by limited proteolysis with trypsin and the peptides produced by cyanogen bromide treatment. Molec. Immun. 18, 949-959. Segrest J. P. & Jackson R. L. (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Meth. Enzym. 28, 54-63. Shaoiro S. S. & Waueh D. F. (1966) The purification of himan prothrombL. Thromb. Diath. haemorrh. 16, 469-490. Weber K. & Osborn M. (1969) The reliability of molecular weight determinations by sodium dodecyl sulfate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. Yphantis D. A. (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3, 297-3 17.