A protease-sensitive site in the proposed Ca2+-binding region of

Protein Science (1992), I , 700-709. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society

A protease-sensitive site in the proposed Ca2+-binding region of human serum amyloid P component and other pentraxins

CAROL M. KINOSHITA,'s4 ANITA T. GEWURZ,' JOAN N. SIRGEL,'

ROBERT HUCKMAN,' AND HENRY GEWURZ' ' Department of lmmunology/Microbiology, Rush Medical College, Chicago, Illinois 60612 ' Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Rocky Mountain Laboratories, National Institutes of Health, Hamilton, Montana 59840

SHAN-CHING YING,' TONY E. H U G L I , ~ JOHN E. C O E , ~ RAJESH K. GUPTA,'

(RECEIVED November 1, 1991; REVISED MANUSCRIPT RECEIVED February 5, 1991)

Abstract

Serum amyloid P component (SAP) is a decamer of 10 identical 25.5-kDa subunits. Limited proteolysis of SAP with a-chymotrypsin cleaves the subunit into two fragments of 18 and 7.5 kDa, although the fragments stay together in the decamer under nondenaturing conditions. Proteolysis does not occur in the presence of CaZ+ (10 mM). Cleavage with a-chymotrypsin prevents the Ca2+-dependent binding of SAP to zymosan extract, nucleosomes, and DNA. The a-chymotrypsin cleavage site identified is in a region of SAP that is highly conserved in members of the human C-reactive protein (CRP) family of proteins (pentraxins) to which SAP belongs and is similar to the Ca2+-binding site in calmodulin and related Caz+-binding proteins (Nguyen, N.Y., Suzuki, A., Boykins, R.A., & Liu, T.-Y., 1986, J. Biol. Chem. 261, 10456-10465). Treatment of SAP with other proteases (trypsin, Pronase, and Nagarse protease) yields fragmentation patterns upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that are similar to those obtained with a-chymotrypsin. Two other members of the pentraxin family of proteins, hamster female protein and rabbit CRP, also exhibit similar fragmentation patterns on SDS-PAGE when treated with the various proteases. Recently, it has been shown that the homologous protein, human CRP, is cleaved in the same homologous position as cleavage of SAP by a-chymotrypsin, resulting in the loss of Ca2+ binding (as shown by equilibrium dialysis) and Caz+-dependent binding reactivities (Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H., 1989, Biochemistry 28,9840-9848). These results indicate that the protease sensitivity of this proposed CaZ+- binding region has been conserved and may play an important regulatory role, perhaps via the control of Ca2+- dependent properties of these proteins.

Keywords: calcium-binding protein; C-reactive protein; hamster female protein; pentraxin; serum amyloid P component

Amyloid P component is a glycoprotein composed of 10 identical 25.5-kDa subunits arranged as two cyclic pen- tamers stacked face to face (Pinteric et al., 1976; Pinteric & Painter, 1979). Amyloid P component is present in serum (Binette et al., 1974; Pepys et al., 1978), amyloid deposits in tissues (Cathcart et al., 1965, 1967a,b; Binette et al., 1974; Skinner et al., 1974; Glenner, 1980; Ander-

Reprint requests to: Henry Gewurz, Department of Immunology/Mi-

Present address: Department of Biology, Massachusetts Institute crobiology, Rush Medical College, Chicago, Illinois 60612.

of Technology, Cambridge, Massachusetts 02139.

son & Mole, 1982; Mantzouranis et al., 1985; Prelli et al., 1985), normal glomerular basement membrane covalently associated with collagen (Dyck et al., 1980a,b), and elastic fibers in skin and blood vessels (Breathnach et al., 1981). In the presence of Ca2+, human serum amyloid P component (SAP) binds to the yeast cell wall extract, zymosan (Pepys et al., 1979; Potempa et al., 1985), the complement component Clq (Bristow & Boackle, 1986), chromatin (Pepys & Butler, 1987; Gewurz et al., 1990), DNA and H1 histone (Pepys & Butler, 1987; Butler et al., 1990), agarose (Hind et al., 1984), heparan and dermatan sulfate (Hamazaki, 1987), fibronectin (Rostagno et al.,

700

Protease-sensitive Ca2+-binding region in pentraxins

1986), and modified C-reactive protein (CRP) (Harris et al., 1989). A function for amyloid P in the body has not been established, although Pepys and Butler (1987) found that SAP is the major component in serum that binds to DNA.

SAP is a member of the CRP family of proteins, which shares sequence homology and is generally configured as a cyclic pentamer, hence the name, pentraxin (Osmand et al., 1977). Ca2+-dependent binding to various ligands is another characteristic conserved in all members of the CRP family. A highly conserved region resides between residues 133 and 147 of human CRP and residues 13 1 and 145 of human SAP, which displays sequence similarities to the Ca2+-binding site in calmodulin and related proteins (Nguyen et al., 1986a,b; Swanson et al., 1991). Direct evidence for the binding of Ca2+ to this site in human CRP has been presented elsewhere (Kinoshita et al., 1989). The present work extends these findings to human SAP and suggests that other pentraxins (rabbit CRP and hamster female protein) may also exhibit protease-sensitive cleavage in the proposed Ca2+-binding region.

Results

Cleavage of SAP with various proteases

SAP was treated with various proteases and subjected to gel filtration fast performance liquid chromatography (FPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of SAP with a - chymotrypsin, trypsin, Pronase, or Nagarse protease did not substantially affect the elution behavior of SAP from Superose 12 in physiological buffers. Elution profiles of untreated and a-chymotrypsin-treated SAP are shown in Figure 1. SAP treated with Pronase or Nagarse protease exhibit elution profiles similar to that obtained with a - chymotrypsin (data not shown). The peak material eluting at or near the position of the intact SAP decamer was collected from each digest for further analysis. Superose 12 FPLC serves to separate SAP from the proteases because proteases elute after the decamer peak, as determined by chromatography of each protease alone (not shown).

SDS-PAGE patterns of a-chymotrypsin, trypsin, and Pronase digests of SAP are shown in the presence and absence of 10 mM CaC1, (Fig. 2). The SDS gel of the Nagarse protease digest is not shown but the pattern looks very similar to that shown for Pronase. All proteases gen- erate fragments of the SAP subunit that are between 65 and 70% the size of the intact subunit. A <14-kDa fragment of SAP is also visible in the gels of the a-chymotrypsin and Pronase digests. CaCl, at 10 mM protects SAP from proteolysis by all of the proteases tested. Re- duction of the disulfide bonds in the samples with dithiothreitol prior to SDS-PAGE does not appreciably affect

0.9

0.6

0.3

0 a0

a

0.6

0.3

0

70 1

10 15 20 25

Elut ion Volume (ml) Fig. 1. Superose 12 gel filtration FPLC (in nondenaturing buffer) of SAP treated with a-chymotrypsin. The column buffer was 10 mM Tris/NaCI, containing I mM EDTA. SAP was incubated alone for 3 h at 37 "C (A) and with a-chymotrypsin for 3 h at 37 "C (B).

the apparent size of the material in the bands (not shown).

Gel filtration and SDS-PAGE of a-chymotrypsin-treated SAP

The degradation of SAP by a-chymotrypsin was chosen for further study. a-Chymotrypsin produces a clear and simple fragmentation of SAP based on analysis by SDS- PAGE. The intact SAP subunit migrates with an apparent M, of 31 kDa upon SDS-PAGE, which is somewhat larger than the actual value of 25.5 kDa. Treatment of SAP with a-chymotrypsin in the absence of CaC12 gen- erates two fragments on SDS gels. Fragment A is 66% the size of the intact subunit, and fragment B is too small (<14 kDa) to be sized in this gel system (Fig. 3, lane 2). The extent of fragmentation is greatest when digestion is carried out in the absence of CaCl, or in the presence of submillimolar CaCl, (Fig. 3, lanes 1 and 2). A concentration of 1 mM CaCI, partially protects SAP from

702

Intact

A .

B ,

1 2 3 4 5 6 7 8 9 k Da

- 97

-66

-43

- 31

- 21

-14

Fig. 2. SDS-PAGE of SAP digested with various proteases in the presence or absence of 10 mM CaCI2. Lane 1 shows SAP incubated alone for 3 h at 37 "C in the presence of 10 mM added CaCI,; lane 2 shows SAP incubated alone in the absence of CaCI,; lane 3 shows SAP incubated with Pronase in the presence of 10 mM CaCI2; lane 4 shows SAP incubated with Pronase in the absence of CaCI,; lane 5 shows SAP incubated with trypsin in the presence of CaCI,; lane 6 shows SAP incubated with trypsin in the absence of I O mM CaCI,; lane 7 shows SAP incubated with a-chymotrypsin in the presence of I O mM CaCI,; lane 8 shows SAP incubated with a-chymotrypsin in the absence of CaC1,; and lane 9 shows the BioRad standard.

digestion (Fig. 3, lane 3), and 10 mM CaC12 confers complete protection from proteolysis (Fig. 3, lane 4). The presence of dithiothreitol in the SDS gel sample does not affect the apparent M , of the fragments (not shown).

Gel filtration of SAP digest under denaturing conditions

Gel filtration FPLC of SAP was performed in denaturing agent (i.e., 6 M guanidine-HCI) (1) to confirm the M, of fragment A; (2) to determine the approximate M, of fragment B; and (3) to separate the fragments so that purified fragments could be further analyzed. The Su- perose 12 FPLC elution profile of the a-chymotrypsin digest of SAP performed in 50 mM Tris at pH 8.6 and containing 6 M guanidine-HC1 (Tridguanidine) is shown in Figure 4. Three major peaks were observed at apparent M,'S of 25 kDa, 18 kDa, and 9 kDa. By SDS-PAGE, it was found that peak 1 corresponds to the intact SAP subunit, peak 2 corresponds to fragment A, and peak 3 corresponds to fragment B (gel data not shown).

Characterization of SAP fragments

The sequence of the first 10 residues of the polypeptide in fragment B (the 9-kDa fragment) was determined and coincides with residues 145-154 of SAP (Fig. 5). The apparent M, of fragment B, as determined by FPLC elu-

Intact-

A-

C.M. Kinoshita et al.

1 2 3 4 5 6

kDa

. -97 P -66

-43

-31

- 21

-14

B-

Fig. 3. SDS-PAGE of SAP digested with a-chymotrypsin in the presence of various concentrations of CaCI,. SAP was incubated in the various CaCl, buffers with 10% (w/w) a-chymotrypsin for 3 h at 37 "C. Prior to electrophoresis, the digests were fractionated on the Superose 12 FPLC column in 10 mM Tris containing 0.15 M NaCI, pH 7.3, to remove a-chymotrypsin. Material in the peak eluting at the position of intact SAP was collected and subjected to SDS-PAGE. Lane 1 shows SAP incubated with a-chymotrypsin in the absence of added CaCI,; lanes 2, 3, and 4 show SAP incubated with a-chymotrypsin in the presence of 0.1 mM, 1 mM, and IO mM added CaCI,, respectively; lane 5 shows SAP incubated without protease; and lane 6 shows the BioRad standard.

0.4

0.3

g 0.2

a (v

0.1

I 1 1

(D

Y v) hl

n

6 14 22

Elution Volume (mi) Fig. 4. Superose 12 gel filtration FPLC (in denaturing buffer) of SAP digested with a-chymotrypsin. The column buffer was 50 mM Tris-CI, containing 6 M guanidine hydrochloride, pH 8.6.

Protease-sensitive Ca2+-binding region in pentraxins 703

t F r a g m e n t A (1-144)

F r a g m e n t B (145-204)

131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154

- 1 - V - L - G - Q - E - Q - D - S - Y - G - G - K - F - D - R - S - Q - S - F - V - G - E - I - b k I

a-Chymotrypsin

Fig. 5. Schematic representation of human SAP indicating the position of cleavage by a-chymotrypsin and the resultant fragments A and B, the residues identified upon partial amino acid sequenation of fragment B (residues 145-154), and the proposed Ca2+-binding region (residues 136-147, box) as discussed in the text.

tion in guanidine, approximates the theoretical M, of a polypeptide composed of residues 145 to the carboxyl- terminal end of the subunit (residue 205). This theoretical M, is 7.4 kDa, assuming 11 070 carbohydrate (Thomp- son & Enfield, 1978). a-Chymotrypsin cleaves carboxyl to hydrophobic residues. Thus, cleavage between residues 144 and 145 is consistent with the specificity of a-chymotrypsin, as residue 144 is Phe.

A single cleavage between residues 144 and 145 would result in a large fragment composed of residues 1-144. Cleavage by a-chymotrypsin also could occur between residues 140 and 141 or between residues 123 and 124, resulting in fragments composed of residues 1-140 or 1-123, respectively. However, the observed amino acid composition of fragment A is the best match for the composition of residues 1-144 (Table 1). The apparent M, of fragment A by FPLC in Tridguanidine is 18 kDa; this is consistent with the theoretical M, of 17.6 kDa for residues 1-144, assuming 11 Yo carbohydrate by weight (Thompson & Enfield, 1978). The theoretical M, of a fragment composed of residues 1-123 is approximately 15 kDa, which is substantially smaller than the observed value of 18 kDa.

Effect of cleavage on binding of SAP to zymosan extract, nucleosomes, and DNA

Binding of SAP to zymosan extract, nucleosomes, and DNA represents the Caz+-dependent properties of SAP. Solid-phase ligand-binding assays were performed to de-

termine whether the specific cleavage of SAP by a-chymotrypsin in the proposed Ca2+-binding region destroys the ability of SAP to bind to these ligands. a-Chymotryp- sin-treated SAP exhibits markedly decreased binding to zymosan extract (Fig. 6A), nucleosomes (Fig. 6B), and DNA (Fig. 6C). The residual binding reactivity remaining can be accounted for by the residual (less than 10%) intact SAP subunit remaining after a-chymotrypsin treatment.

Table 1. Amino acid composition of the large fragment of a-chymotrypsin-treated SAP __ ~

~ -. ~ ~ ~- _ ~ _ _ _

Amino acid Theoretical Experimental

Asx 10 11.0 Thr 7 1.2 Ser 12 12.8 Glx 21 18.4 Pro 8 1.5 Glya 11 11.0 Ala 5 5.9 Val 13 13.2 Ile 7 6.5 Leu 13 14.0 Tyr 8 5 . 5 P he 9 8.7 His 4 3.9 LYS 10 9.3

7 6.8

~~ ." " - ~ ~- - - ~- ~-

"_ ~~~ ~ ~ ~~ _" "" ~ -~

a Normalized to Gly.

704

0.2

0 0.6

0.4

0

Amount SAP (pg/well)

Fig. 6. Binding of intact and a-chymotrypsin-treated biotinylated SAP to immobilized zymosan extract (A), nucleosomes (B), and DNA (C). Intact SAP in CaCI2 (o-o), intact SAP in EDTA (@-@I), and a- chymotrypsin-treated SAP in CaClz (0. . . 0).

Proteolysis of the homologous pentraxins, rabbit CRP, and hamster female protein

Treatment of rabbit CRP and hamster female protein with various proteases did not appreciably affect the native gel filtration FPLC elution profiles of these pentraxins (not shown). SDS-PAGE of the pentraxin digest, after elution from the FPLC column, demonstrates that fragmentation patterns similar to those generated with human CRP are produced. a-Chymotrypsin and trypsin produce little or no fragmentation of rabbit CRP under the conditions used (not shown). Nagarse protease and Pronase digestion each produce a large fragment, which is about 65% the size of the intact subunit and a <14- kDa fragment (Fig. 7, lanes 3 and 4). Figure 8 demonstrates that hamster female protein is also cleaved by

kDa

97-

66-

43-

31 -

1 2 3 4

-Intact

21 -

14- -A

Fig. 7. SDS-PAGE of rabbit CRP treated with various proteases. Prior to electrophoresis, all digests were fractionated on the Superose 12 FPLC column in 10 mM Tris containing 0.15 M NaCl to remove protease. Ma- terial in the peak eluting at the position of intact pentraxin was collected and subjected to SDS-PAGE. Rabbit CRP was incubated for 16 h at 23 "C without protease in lane 2, with Nagarse protease(l0To w/w) for 3 h at 37 "C in lane 3, and with 3% Pronase for 1 h at 23 "C in lane 4; the BioRad standard is shown in lane I .

Pronase, Nagarse protease, and a-chymotrypsin and is not proteolyzed by trypsin. Nagarse protease treatment of hamster female protein produces two large fragments that are 64 and 67% the size of the intact subunit (lane 3), a-chymotrypsin treatment produces a large fragment that is 72% the size of the intact subunit (lane 4), and Pronase treatment produces two large fragments that are 69 and 70% the size of the intact subunit (lane 5) .

Discussion

Human CRP has been found to contain a proteolytically sensitive region near the Ca2+-binding site (Kinoshita et al., 1989). To determine whether this protease-sensitive region exists in other pentraxins, human SAP, hamster female protein, and rabbit CRP were treated with Pro- nase, Nagarse protease, a-chymotrypsin, and trypsin. As with human CRP, after proteolysis, fragments of SAP did not separate in physiological buffers but were observed upon PAGE in SDS. The patterns of these pentraxins in SDS-PAGE gels after proteolysis were similar

Protease-sensitive Ca2+-binding region in pentraxins

1 2 3 kDa

97- - 66- - 43- - 31- - " 21- -

4 5 6

-Intact

"

14-

Fig. 8. SDS-PAGE of hamster female protein treated with various proteases. Samples were prepared as described in the legend to Figure 7. Hamster female protein was incubated without protease for 16 h at 37 "C in lane 6; with Pronase for 1 h at 37 "C in lane 5; with a-chymotrypsin for 16 h at 23 "C in lane 4; with Nagarse protease for 16 h at 37 "C in lane 3; and with trypsin for 16 h at 37 "C in lane 2. The BioRad standard is shown in lane 1.

to those seen after proteolysis of human CRP (Kinoshita et al., 1989), although there were differences in the susceptibility of pentraxins to the various proteases. SAP was sensitive to cleavage by all four proteases, whereas hamster female protein was sensitive to cleavage by Pro- nase, Nagarse protease, and a-chymotrypsin, but not by trypsin. Rabbit CRP was sensitive to cleavage by Pronase and Nagarse protease, only slightly susceptible to a-chymotrypsin, and not susceptible to trypsin. The pattern of susceptibility of rabbit CRP to proteases is similar to that of human CRP, except that human CRP is less sensitive to cleavage by Pronase because a higher ratio of Pronase to CRP is needed to obtain fragmentation of human CRP. The pattern of susceptibility of hamster female protein is intermediate between CRP and human SAP. Whereas hamster female protein is similar to human and rabbit CRP in its resistance to cleavage by trypsin, female protein differs from CRP in that it is more susceptible to cleavage by a-chymotrypsin. When susceptible to cleavage, the pentraxins mentioned above are cleaved such that one or two fragments, which are between 64 and 72% the size of the intact subunit, are produced. An ad- ditional c14-kDa fragment is sometimes visible on the SDS-PAGE gels. Cleavage of these pentraxins occurred one-third of the way in from the carboxy-terminal end of

705

the polypeptide chain. Cleavage at the other end of the polypeptide chain would remain undetected by nonreduc- ing SDS-PAGE because an intrachain disulfide loop is located between residues 36 and 95 in human SAP, 36 and 97 in rabbit CRP, and 35 and 94 in hamster female protein. Reduction of human SAP and rabbit CRP with dithiothreitol does not substantially change the M, of the fragments, indicating that further cleavage within the intrachain disulfide loop does not occur. These results indicate that pentraxins all have a proteolytically sensitive region in approximately the same homologous location, which corresponds to the Caz+-binding site in human CRP. This region is highly conserved in these pentraxins (Nguyen et al., 1986a,b; Swanson et al., 1991), and it ap- pears that protease sensitivity in this region is a characteristic perhaps shared by all pentraxins.

The proteolysis of human SAP with various proteases was examined more closely. The presence of 10 mM CaC12 during protease treatment protects SAP from proteolysis by a-chymotrypsin, Pronase, Nagarse protease, and trypsin (Fig. 2). Proteolysis of SAP by a-chymotrypsin was studied in greater detail. SAP is partially protected from a-chymotrypsin digestion in 1 mM CaCI2 and completely protected in 10 mM CaClz. As reported previously (Kinoshita et al., 1989), human CRP is partially protected from proteolysis by Nagarse protease and Pronase in 0.1 mM CaC12 and completely protected in 1 .O mM CaC12. This indicates that the affinity of human SAP for Ca2+ may be about IO-fold lower than that of human CRP. Furthermore, physiologic concentrations of Ca2+ would be expected to partially protect SAP from proteolysis by these various proteases and completely protect human CRP. Human CRP may depend upon this protease resistance in the acute phase environment where relatively high concentrations of various proteases exist. Protection by Ca2+ has also been reported for plasmin digestion of fibrinogen (Haverkate & Timan, 1977; Mar- guerie, 1977), thrombin and trypsin digestion of thrombospondin (Lawler et al., 1982), and a-chymotrypsin digestion of deoxyribonuclease A (Hugli, 1973) and myosin (Weeds & Pope, 1977).

To determine whether cleavage of SAP by a-chymotrypsin does indeed occur in the same region identified in human CRP (Kinoshita et al., 1989), the fragments generated upon proteolysis of SAP with a-chymotrypsin were characterized. The results demonstrate that cleavage occurs between residues 144 and 145. The homologous position in human CRP is cleaved upon treatment with Pronase (Kinoshita et al., 1989). Thus, cleavage of SAP by a-chymotrypsin occurs in the highly conserved region that is homologous to the Ca2+-binding site in human CRP (Kinoshita et al., 1989).

Cleavage of the SAP subunit in the proposed Ca2+- billding region destroys the ability of SAP to bind to the Ca2+-dependent ligands, zymosan extract, nucleosomes, and DNA (Fig. 6). Calcium may induce a conformational

706

change in SAP, which is conducive to ligand binding, as is the case with calmodulin and related Ca2+-binding proteins (LaPorte et al., 1980; White, 1988). Thus, cleavage by a-chymotrypsin may indirectly affect ligand binding via destruction of the Ca2+-binding site.

Equilibrium dialysis was attempted to determine the affinity and number of Ca2+-binding sites for human SAP. A concentration of about 1 mg/mL of human CRP was required to obtain data from comparable equilibrium dialysis experiments (Kinoshita et al., 1989). Because the affinity of human SAP for Ca2+ seems to be about 10-fold lower than that of human CRP, a concentration of about 10 mg/mL would have been required to obtain data for human SAP. However, SAP tends to precipitate at these high concentrations. Concentrations of up to 4 mg/mL of human SAP were used without success. Thus, the number of Ca2+-binding sites in human SAP remains undetermined.

Comparisons of a highly conserved region in several pentraxins with the 12 key residues of selected Ca2+- binding loops in calmodulin and several related proteins are presented in Table 2 (also see Kinoshita et al., 1989). The major differences between the proposed Ca2+-binding site in the pentraxins and the calmodulin-like sites are that: (1) position 8 in the calmodulin-like Ca2+-binding sites is always occupied by an aliphatic hydrophobic residue, which is not present at the corresponding position in the pentraxins, and (2) position 12 in the calmodulin- like sites is always glutamate, but glutamate occurs in this position in only one pentraxin chain (limulus CRP 1.4). Positions 8-12 of the sites in human CRP (Kinoshita et a]., 1989) and limulus chain 1.4 correspond better with

C.M. Kinoshita et at.

positions 1-5 of the calmodulin-like sites. Thus, the highly conserved Ca2+-binding region in human CRP (Kinoshita et al., 1989) and limulin chain 1.4 may be two half-sites, each corresponding to the amino-terminal half of the calmodulin-like Ca2+ sites. The other half-sites may reside in other parts of the polypeptide chain. In the galactose-binding protein, the last two residues of the loop come from another part of the polypeptide chain, which demonstrates that the entire site need not be se- quential (Vyas et al., 1987). Human SAP and the other pentraxins have neither an asparagine nor acidic side chain at position 8, and therefore residues in positions 8- 12 do not match the consensus sequence for the amino half of the calmodulin-like Ca2+ sites. Thus, there may be only one half-site for Ca2+ binding in the conserved region of human SAP, rabbit CRP, and hamster female protein. Proteolytic cleavage in this region of human SAP destroys Ca2+-dependent ligand binding and may do so via disturbance of the Ca2+-binding site, as ap- pears to be the case with human CRP (Kinoshita et al., 1989).

This work demonstrates that human SAP is cleaved by a-chymotrypsin within a conserved region similar to the Ca2+-binding site in calmodulin and related proteins (Dang et al., 1985; Nyugen et al., 1986a,b; Kinoshita et al., 1989; Swanson et al., 1991). Other members of the CRP family of proteins (rabbit CRP and hamster female protein) also appear to exhibit protease sensitivity in the same region. This suggests that the protease sensitivity of the region is a highly conserved property that may be important in the control of the Ca2+-dependent properties of the CRP family of proteins.

Table 2. Comparison of conserved sequences in pentraxins with the Ca2+-binding regions of known Ca2+-binding loopsa "_ ~ ~~~

~ . . _ _ . _ _ _ ~ ~ . ~ _ _ _ _ _ _ _ ~ _ _ _ _ _ ~ ~ ~ ~ ~~~ ~-

Calcium-binding loop Residue numbers 1 2 3 4 S 6 7 8 9 1 0 1 1 1 2

- .~ ~ ~ _ _ ~ ~ ~ -

~ .~ ~ _ _ ~~ ~ __ - ~~

Calmodulin loop 1 20-3 1 Parvalbumin loop CD 5 1-62 Troponin C loop 1 30-41 Galactose-binding

protein 134-205

Limulus CRP 3.3' 139-155 Limulus CRP 1.1 139-IS5 Limulus CRP 1.4 139-IS5 Human CRP 133-148 Rabbit CRP 132-148 Human SAP 131-147 Hamster FP 131-147

~.

.__ _ _ . ~ ~ ~ _ _

D K D G D Q D K D A D G

D L N K

V V L G Q D Q D S V V L G Q D Q D S V V L G Q E Q D S I I L G Q E Q D S 1 l L G Q D Q D s I V L G Q E Q D S I I L G Q E Q D K

~

" ~~~~ _ _ _ _ ~

N G T I T T K E S G F I E E D E G G D I S T K E

V G G K F D V G G D F D V G G E Y D F G G N F E F G G S F E Y G G K F D Y G G G F D

A T A G A E G S K Q R S N Y

a Includes calmodulin loop 1 (Watterson et al., 1980), parvalbumin loop CD (Coffee & Bradshaw, 1973), troponin C loop 1 (Wilkinson, 1976), and galactose-binding protein (Mahoney et al., 1981).

Residue 204 of the galactose-binding protein. The highly conserved region of the pentraxins identified and related to Caz+-binding by Nguyen et al. (1986a,b) is shown in the box: the res-

idues labeled 1-12 are compared to the Caz+-binding site in calmodulin and related proteins (see Discussion).


Materials and methods

Preparation of human SAP, hamster female protein, and rabbit CRP

SAP was prepared from out-dated plasma obtained from the American Red Cross using a modification of the procedure of deBeer and Pepys (1982) as described by Potempa et al. (1985). Purification of rabbit CRP was carried out as described by Cabana et al. (1982), and hamster female protein was prepared as described by Coe et al. (1981).

Limited proteolysis of SAP

SAP at 1-2 mg/mL in 50 mM Tris-C1, pH 7.3, containing 0.15 M NaCl, was digested with 10% (w/w, protease/ substrate) a-chymotrypsin, trypsin, Pronase, or Nagarse protease for 3 h at 37 "C. The digests were chromatographed on a 1 x 30-cm Superose 12 gel filtration FPLC column, equilibrated and eluted with Tris/NaCl (10 mM Tris-C1, pH 7.3, 0.15 M NaCl) containing 1 mM EDTA at a flow rate of 0.3 mL/min. Absorbance at 280 nm (A280) of the eluate was continuously monitored, and the material in the peaks was collected. BioRad gel filtration standard, containing thyroglobulin (670 kDa), immuno- globulin G (158 kDa), ovalbumin (45 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa), was used for mo- lecular mass (Mr) calibration of the FPLC column.

Effect of Caz+ on susceptibility of SAP to proteolysis

To test the effect of Ca2+ on susceptibility to digestion by proteases, SAP was preincubated for 1 h at room temperature in Tris/NaCI containing 0, 0.1 mM, 1 .O mM, or 10 mM added CaC12. Protease was added to 10% (w/w), and the mixtures were incubated for 3 h at 37 "C. The digests were immediately chromatographed on the Superose 12 column as described above for limited proteolysis, and the material in the peaks was collected.

Protein assay

Protein concentration was determined by the BCA protein assay (Pierce Chemical Co.; Smith et al., 1985).

SDS-PAGE

SDS-PAGE on 13% polyacrylamide gels was carried out on the material in the peaks from the FPLC column according to the method of Laemmli (1970). Dithiothreitol to 0.1 M final concentration was added to some samples before heating to reduce disulfide bonds. BioRad low M, SDS-PAGE standard was used for M, calibration.

Separation of fragments by gel filtration in denaturing buffer

Protease digestion conditions were modified as follows in preparation for gel filtration in denaturing buffer. SAP at 1-2 mg/mL was mixed with 1 Vo (w/w) a-chymotrypsin and incubated for 1.5 h at 37 "C, and another 1 Yo (w/w) a-chymotrypsin was added. After 3 h total time of incubation, 1 Vo (w/w) a-chymotrypsin again was added, and the digestion was quenched after 4.5 h total incubation time with 0.5 mM (final concentration) phenylmethylsul- fonyl fluoride (PMSF). Solid guanidine hydrochloride was added to the quenched digest to a concentration of 4 M, and the digest was chromatographed on the Superose 12 FPLC column, and equilibrated and eluted with 50 mM Tris-C1, pH 8.6, containing 6.0 M guanidine hydrochloride. The flow rate was 0.05 mL/min and the A280 was continuously monitored. Sample peaks were pooled and the material was dialyzed in Spectrapor dialysis tubing (3.5 kDa cutoff) on a magnetic stirrer against one change of 4 L of 0.1 M ammonium carbonate, pH 8.6, at 4 "C. The dialyzed material was lyophilized and subjected to SDS-PAGE, amino acid analysis, or partial sequencing. Carbonic anhydrase (29 kDa), myoglobin (17 kDa), cy- tochrome C (12.5 kDa), and aprotinin (6.5 kDa) were used for M, calibration of the column.

Amino acid analysis and sequencing

Amino acid analyses and protein sequencing were performed by the Protein Analysis Service at Scripps Clinic and Research Foundation (La Jolla, California). Amino acid analysis was performed on a Beckman 6900 Auto- analyzer. Protein samples of 5-20 pg were placed in igni- tion tubes, vacuum sealed, and hydrolyzed at 110 "C for 24 h in constant-boiling 6 M HCI, containing 1% (v/v) phenol. For partial amino acid sequencing, automated Edman degradation of SAP fragments was performed on an Applied Biosystems Model 470A Sequenator. The phenylthiohydantoin (PTH) amino acid derivatives were identified by HPLC analysis using a Waters automatic sample injection and chromatographic system fitted with a Zorbax 5-pm PTH column (4.6 x 220 mm).

Biotinylation of SAP

SAP at 1-2 mg/mL in 50 mM Tris-C1, containing 0.15 M NaCl and 5 mM CaClz, pH 7.3, was mixed with 1 volume of 1 mg/mL NHS-LC-biotin (Pierce Chemical Co.) and incubated for 4 h at 23 "C. The mixture was then dialyzed on a magnetic stirrer against one change of 4 L TridNaC1.

Preparation of nucleosomes

Chicken erythrocyte nuclei were prepared according to methods described by Weintraub et al. (1975) and Shaw

708 C.M. Kinoshita et ai.

et al. (1976). The erythrocytes were lysed by suspension in hypotonic buffer consisting of 10 mM Tris-HCI, containing 0.5% Triton X-100, 10 mM NaCl, 5 mM MgC12, 0.25 mM PMSF (pH 7.4), and centrifuged at 3,000 x g for 10 min. The nuclear pellet was washed four times with the Triton buffer and once with 10 mM Tris-HC1, pH 7.4, containing 75 mM NaC1,0.2 mM EDTA, and 0.1 mM PMSF. The nuclear pellet was suspended in 10 mM Tris-C1, pH 8.0, containing 250 mM sucrose and 1 mM CaC1, such that the absorbance at AZm was equivalent to 100. The suspension was digested with 200 units/mL micro- coccal nuclease at 37 "C for 30 min, and the digestion was quenched by the addition of EDTA to 20 mM. The digest was cooled to 0 "C and centrifuged at 3,000 x g for 10 min. The nuclear pellet was resuspended in 0.25 mM EDTA, pH 7.0, using a volume half that of the original suspension and dialyzed for 16 h against the same buffer. The dialysate was centrifuged at 3,000 x g for 15 min, and the supernatant was adjusted to an A260 of 100 and fractionated on an isokinetic sucrose gradient (Cm = 5 % , particle density 1.51 g/cm3 at 4 "C; McCarty et al., 1974) containing 25 mM Tris-HC1, pH 8.0, and 1 mM EDTA for 20-22 h at 25,000 rpm and 4 "C in a Beckman SW27 rotor. Gradients were emptied by pumping from the bottom of the tube, and 1-mL fractions were collected. The second peak contained the greatest amount of material as determined by A260 and was used for solid phase assays.

Preparation of zymosan extract

Zymosan extract was prepared as described by Kubak et al. (1988).

Solid-phase ligand-binding assays

Wells of microtiter plates were coated with 100 pL of a 1 : 1 ,OOO dilution of zymosan extract, 20 pg/mL chicken erythrocyte nucleosomes, or 20 pg/mL DNA (calf thymus DNA, Sigma Chemical Co.), each in 50 mM sodium carbonate, pH 8.6, for 16 h at room temperature. The wells were washed four times using a Nunc-Immuno Wash 8 with 50 mM veronal, containing 0.15 M NaCl, 0.05% Tween 20, and either 2 mM CaCl, or 10 mM EDTA, pH 7.5. Bovine serum albumin (200 pL or 1% [w/v] in H20) was applied to each well. The plates were incubated at 37 "C for 30 min, and the wells were washed. Samples (100 pL) of biotinylated SAP in 2 mM CaC1, or 10 mM EDTA were applied to the wells and incubated for 30 min at 37 "C. The wells were washed, and 100 pL of avidin- peroxidase (18,000-fold dilution in wash buffer) were applied. The plates were incubated for 30 min at 37 "C and washed. Substrate (100 pL of 2,2"azinobis(ethylbenzothi- azolinesulfonic acid) diammonium salt in 0.1 M sodium citrate at pH 4.0) was applied and allowed to develop at

room temperature for 15-60 min. A414 was read in a Titertek Multiskan MC plate reader.

Limited proteolysis of hamster female protein and rabbit CRP

Hamster female protein and rabbit CRP were dialyzed against one change of 4 L of Tris/NaCI at 4 "C. The proteins at approximately 1 mg/mL were treated with proteases as described in the legends to Figures 7 and 8. Rabbit CRP at approximately 1 mg/mL was also treated with trypsin and a-chymotrypsin as described above for SAP. Gel filtration FPLC in Tris/NaCl and SDS-PAGE were carried out as described above for SAP.

Acknowledgments

The excellent technical assistance of Putrina Dunlap, Michael Chen, and Michele Halberg is much appreciated. This work was presented in part at the Annual Meetings of the American As- sociation of Immunologists (Kinoshita & Gewurz, 1987). H.G. is the holder of the Thomas J. Coogan, Sr., Chair in Immunol- ogy established by Marjorie Everett Lindheimer.

References Anderson, J.K. & Mole, J.M. (1982). Large scale isolation and partial

primary structure of human plasma amyloid P-component. Ann. N. Y. Acad. Sci. 389, 216-234.

Binette, P., Binette, M., & Calkins, E. (1974). The isolation and iden-

rhem. J. 143, 253-254. tification of the P-component of normal human plasma protein. Bio-

Breathnach, S.M., Melrose, S.M., Bhogal, B., deBeer, F.C., Dyck, R.F., Tennent, G., Black, M.M., & Pepys, M.B. (1981). Amyloid P component is located on elastic fibre microfibrils in normal human tissue. Nature 293, 652-653.

Bristow, C.L. & Boackle, R.J. (1986). Evidence for the binding of human serum amyloid P component to Clq and Fab. Mol. Immunol.

Butler, P.J.G., Tennent, G.A., & Pepys, M.B. (1990). Pentraxin-chromatin interactions: Serum amyloid P component specifically dis- places H1-type histones and solubilizes native long chromatin. J. Exp. Med. 172, 13-18.

Cabana, V.G., Gewurz, H., & Siegel, J.N. (1982). Interaction of very low density lipoproteins (VLDL) with rabbit C-reactive protein. J . Immunol. 128, 2342-2348.

Cathcart, E.S., Comerford, F.R., & Cohen, A.S. (1965). Immunologic studies on a protein extracted from human secondary amyloid. N. Engl. J. Med. 273, 143-146.

Cathcart, E.S., Shirahama, T., & Cohen, A.S. (1967a). Isolation and identification of a plasma component of amyloid. Biochim. Biophys. Acta 147, 392-393.

Cathcart, E.S., Wolheim, F.A., & Cohen, A S . (1967b). Plasma protein constituents of amyloid fibrils. J. Immunol. 99, 376-385.

Coe, J.E., Margossian, S.S., Slayter, H.S., & Sohn, J.A. (1981). Ham- ster female protein. A new pentraxin structurally and functionally similar to C-reactive protein and amyloid P component. J. Exp. Med. 153, 977-991.

Coffee, C.J. & Bradshaw, R.A. (1973). Carp muscle calcium-binding protein. I . Characterization of tryptic peptides and the complete amino acid sequence of component B. J. Bid. Chem. 248, 3302-3312.

Dang, C.V., Ebert, R.F., & Bell, W.R. (1985). Localization of a fibrinogen calcium binding site between gamma-subunit position 3 I 1 and 336 by terbium fluorescence. J. Biol. Chem. 260, 9713-9719.

deBeer, F.C. & Pepys, M.B. (1982). Isolation of human C-reactive pro-

23, 1045-1052.


tein and serum amyloid P component. J. Immunol. Methods 50,

Dyck, R.F., Evans, D.J., Lockwood, C.M., Rees, A.J., Turner, D., & Pepys, M.B. (1980a). Amyloid P-component in human glomerular basement membrane. Abnormal patterns of immunofluorescent staining in glomerular disease. Lancet ii, 606-609.

Dyck, R.F., Lockwood, C.M., Kershaw, M., McHugh, N., Duance, V.C., Baltz, M.L., & Pepys, M.B. (1980b). Amyloid P-component is a constituent of normal human glomerular basement membrane. J. Exp. Med. 152, 1162-1174.

Gewurz, A., Siegel, J.N., Ying, S.-C., Rite, F., & Gewurz, H. (1990).

FASEB J. 4, A291 1. Monoclonal antibodies reactive with human amyloid P component.

Glenner, G.G. (1980). Amyloid deposits and amyloidosis. The beta- fibrilloses (first of two parts). N. Engl. J. Med. 302, 1283-1292.

Hamazaki, H. (1987). Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate. J. Bioi. Chem. 262, 1456-1460.

Harris, D.E., Gewurz, H., Konoshita, C., Taylor, S., Eatman, J., & Gewurz, A.T. (1989). Reactivity of serum amyloid P component with C-reactive protein and IgM. Clin. Res. 37, 614A.

Haverkate, F. & Timan, G. (1977). Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragment D. Thromb. Res. 10, 803-812.

Hind, C.R.K., Collins, P.M., Rem, K., Cook, R.B., Caspi, C., Baltz, M.L., & Pepys, M.B. (1984). Binding specificity of serum amyloid P-component for the pyruvate acetal of galactose. J. Exp. Med. 159,

Hugli, T.E. (1973). The preparation and characterization of an active derivative of bovine pancreatic deoxyribonuclease A formed by se- lective cleavage with alpha-chymotrypsin. J. Bioi. Chem. 248,

Kinoshita, C.M. & Gewurz, H. (1987). Proteolysis of C-reactive protein and amyloid P component by various proteases. Fed. Proc. 46, 397.

Kinoshita, C.M., Ying, S.-C., Hugli, T.E., Siegel, J.N., Potempa, L.A., Jiang, H.J., Houghten, R.A., & Gewurz, H. (1989). Elucidation of a protease-sensitive site involved in the binding of calcium to C-reactive protein. Biochemistry 28, 9840-9848.

Kubak, B.M., Potempa, L.A., Anderson, B., Mahklouf, S., Venegas, M., Gewurz, H., & Gewurz, A. (1988). Evidence that serum amyloid P component binds to mannose-terminated sequences of poly- saccharides and glycoproteins. Mol. Immunol. 25, 851-858.

Laemmli, U.K. (1970). Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 227, 680-685.

LaPorte, D.C., Wierman, B.M., & Storm, D.R. (1980). Calcium-induced exposure of a hydrophobic surface on calmodulin. Biochemistry 19,

Lawler, J., Chao, F.C., & Cohen, C.M. (1982). Evidence for calcium- sensitive structure in platelet thrombospondin. Isolation and partial characterization of thrombospondin in the presence of calcium. J. Bioi. Chem. 20, 12257-12265.

Mahoney, W.C., Hogg, R.W., & Hermodson, M.A. (1981). The amino acid sequence of the D-galactose-binding protein from Escherichia coli B/r. J. Bioi. Chem. 256, 4350-4356.

Mantzouranis, E.C., Dowton, S.B., Whitehead, AS. , Edge, M.D., Bruns, G.A.P., & Colten, H.R. (1985). Human serum amyloid P component. cDNA isolation, complete sequence of pre-serum amyloid P component, and localization of the gene to chromosome 1. J. Bioi. Chem. 260, 7752-7756.

Marguerie, G. (1977). The binding of calcium to fibrinogen: Some structural features. Biochim. Biophys. Acta 494, 172-181.

McCarty, K.S., Jr., Vollmer, R.T., & McCarty, K.S. (1974). Improved computer program data for the resolution and fractionation of mac- romolecules by isokinetic sucrose density gradient sedimentation. Anal. Biochem. 61, 165-183.

Nguyen, N.Y., Suzuki, A,, Boykins, R.A., & Liu, T.-Y. (1986a). The

261, 10456-10465. amino acid sequence of Limulus C-reactive protein. J. Bioi. Chem.

17-31.

1058-1069.

1712-1718.

3814-3819.

Nguyen, N.Y., Suzuki, A., Cheng, S.-M., Zon, G., & Liu, T.-Y. (1986b). Isolation and characterization of limulus C-reactive protein genes. J. Bioi. Chem. 261, 10450-10455.

Osmand, A.P., Friedenson, B., Gewurz, H., Painter, R.H., Hofmann, T., & Shelton, E. (1977). Characterization of C-reactive protein and the complement subcomponent Clt as homologous proteins display- ing cyclic pentameric symmetry (pentraxins). Proc. Natl. Acad. Sci.

Pepys, M.B. & Butler, P.J.G. (1987). Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum. Biochem. Biophys. Res. Commun. 148, 308-313.

Pepys, M.B., Dash, A.C., Markham, R.E., Thomas, H.C., Williams, B.D., & Petrie, A. (1978). Comparative clinical study of protein SAP (amyloid P component) and C-reactive protein in serum. Clin. Exp. Immunol. 32, 119-124.

Pepys, M.B., Dyck, R.F., deBeer, F.C., Skinner, M., & Cohen, A S . (1979). Binding of serum amyloid P component (SAP) by amyloid fibrils. Clin. Exp. Immunol. 38, 284-293.

Pinteric, L., Assimeh, S.N., Kells, D.I.C., & Painter, R.H. (1976). The ultrastructure of Clt , a subcomponent of the first component of complement: An electron microscopic and ultracentrifuge study. J. Immunol. I 17, 79-83.

Pinteric, L. &Painter, R.H. (1979). Electron microscopy of serum amyloid protein in the presence of calcium; alternative forms of assem- bly of pentagonal molecules in two-dimensional lattices. Can. J. Biochem. 57, 727-736.

Potempa, L.A., Kubak, B.M., & Gewurz, H. (1985). Effect of divalent metal ions and pH upon the binding reactivity of human serum amyloid P component, a C-reactive protein homologue, for zymosan. J. Bioi. Chem. 260, 12142-12147.

Prelli, F., Pras, M., & Frangione, B. (1985). The primary structure of human tissue amyloid P component from a patient with primary id- iopathic amyloidosis. J. Bioi. Chem. 260, 12895-12898.

Rostagno, A., Frangione, B., Pearlstein, E., & Garcia-Pardo, A. (1986). Fibronectin binds to amyloid P component. Localization of the binding site to the 31,000 dalton C-terminal domain. Biochem. Biophys. Res. Commun. 140, 12-20.

Shaw, B.R., Herman, T.M., Kovacic, R.T., Beaudreau, G.S., & Van Holde, K.E. (1976). Analysis of subunit organization in chicken erythrocyte chromatin. Proc. Natl. Acad. Sci. USA 73, 505-509.

Skinner, M., Cohen, A.S., Shirahama, T., & Cathcart, E.S. (1974). P- component (pentagonal unit) of amyloid: Isolation, characterization and sequence analysis. J. Lab. Clin. Med. 84, 604-614.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goede, N.M., Olson, B.J., & Klenk, D.C. (1985). Measurement of protein using bicincho- ninic acid. Anal. Biochem. 150, 76-85.

Swanson, S.J., Mullenix, M.C., & Mortensen, R.F. (1991). Monoclo- nal antibodies to the calcium-binding region peptide of human C- reactive protein alter its conformation. J. Immunol. 147, 2248-2252.

Thompson, A.R. & Enfield, D.L. (1978). Human plasma P component: Isolation and characterization. Biochemistry 17, 4304-431 1.

Vyas, N.K., Vyas, M.N., & Quiocho, EA. (1987). A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis. Nature 327, 635-638.

Watterson, D.M., Sharief, F.S., & Vanaman, T.C. (1980). The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J. Bioi. Chem. 255, 962-971.

Weeds, A.G. & Pope, B. (1977). Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility.

Weintraub, H., Palter, K., & Van Lente, F. (1975). Histones H2a, H2b, H3, and H4 form a tetrameric complex in solutions of high salt. Cell

White, H.D. (1988). Kinetic mechanism of calcium binding to whiting

Wilkinson, J.M. (1976). The amino acid sequence of troponin C from

USA 74, 739-743.

J. Mol. Bioi. 111, 129-157.

6, 85-1 10.

parvalbumin. Biochemistry 27, 3357-3365.

chicken skeletal muscle. FEBS Lett. 70, 254-256.

Documents

A protease-sensitive site in the proposed Ca2+-binding region of