The oligosaccharide chains of cobra venom factor are required for complement activation

Molecular Immunology, Vol. 26, No. 6, pp. 563-574, Printed in Great Britain.

0161-5890/89 $3.00 + 0.00 Pergamon Press plc

1989

THE OLIGOSACCHARTDE CHAINS OF COBRA VENOM FACTOR ARE REQUIRED FOR COMPLEMENT ACTIVATION* ALICE H. GRIER~~

and CARL.-WILHELM

VOGEL$//

Departments of Biochemistry and Medicine, and the Center for Interdisciplinary Research on Immunologic Diseases, Georgetown University School of Medicine, Washington, DC 20007, U.S.A.

(First received 1 September 1988; accepred in revisedform

27 December

1988)

Abstract--To examine the function of the carbohydrate chains of cobra venom factor (CVF), the molecule was enzymatically deglycosylated under non-denaturing conditions with N-glycanase (peptide-N4-(N-acetyl/J-glucosaminyl) asparagine amidase). The deglycosylation of CVF chains seems to proceed independently of each other, leading to partially deglycosylated intermediates. Complete deglycosylation of CVF was found to abolish the activity of CVF. The deglycosylated molecule is unable to activate the alternative

pathway of complement. Deglycosylated CVF no longer consumes the serum complement activity, it does not induce C3 activation in serum, nor does it induce complement-mediated hemolysis. These results indicate that the carbohydrate moieties of CVF are essential for its role in complement activation.

INTRODUCTION

Cobra venom factor (CVF) is a glycoprotein in cobra venom that activates complement in mammalian serum by functioning like C3b, the activated form of C3 (for review see Pangburn and Miiller-Eberhard, 1984). Like C3b, CVF binds to Factor B of the alternative pathway of complement (Hensley et al., 1986). When bound to CVF or C3b, Factor B is cleaved by Factor D into Bb, which remains bound to CVF or C3b, and Ba, which is released (Vogt et al., 1974; Lesavre et af., 1979). The structures of the two bimolecular complexes, CVF,Bb and C3b,Bb, have been elucidated by electron microscopy (Smith et al., 1982, 1984). Both complexes are alternative pathway C3jCS convertases (EC 3.4.21.47). The catalytic site resides in the Bb subunit which cleaves *Part of this work was presented at the XIIth International Complement Workshop, Chamonix, France, September 1987 (Crier and Vogel, 1987). This work was supported by NIH grants CA 355525, HL 29523 and AI 15321 and a pre-doctoral fellowship from Animal Genetic Systems Inc.. Rockville, MD. TPortions of this work were performed by A. H. Crier in partial fulfillment of the requirements for the Ph.D. degree, Department of Biochemistry, Georgetown University. ICurrent address: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, U.S.A. §Author to whom correspondence should be addressed. IIC.-W. Vogel is recipient of Research Career Development Award CA 01039 from the National Cancer Institute, United States Department ofHealth and Human Services. Abbreviations: CVF, cobra venom factor; VBS, Verona1 gelatin, CaCl, buffered saline; GVB 2+, VBS containing and MgCl,; SDS, sodium dodecyl sulfate; NHS, normal human serum. 563

the alpha chain of complement components C3 and C5, respectively. Kinetic and thermodynamic data of C3 cleavage have been reported for both enzymes (Vogel and Miiller-Eberhard, 1982; Pangburn and Miiller-Eberhard, 1986). In addition to the functional similarity of CVF and C3b of forming a C3jCS convertase with Factor B, the two proteins have been shown to share antigenic epitopes. Immunologic cross-reactivity of both pdlyclonal antisera and monoclonal antibodies to CVF with human C3 (Alper and Balavitch, 1976; Minta and Man, 1980; Eggertsen et al., 1983; Vogel et al., 1984; Grier et al., 1987), and of polyclonal antisera and a monoclonal antibody to human C3 with CVF (Grier et al., 1987), have been reported. Many structural similarities between CVF and C3 and its physiological activation products, C3b and C3c, have been found, including amino acid composition, circular dichroism spectra and secondary structure, isoelectric points, electron microscopic ultrastructure and amino-terminal amino acid sequences (Smith et al., 1982, 1984: Vogel et al., 1984; Lundwall et al., 1984a; Vogel and Pangburn, 1985). Based on the number and mol. wts of the polypeptide chains, amino acid compositions, secondary structures and amino-terminal amino acid sequences, it was concluded that CVF structurally resembles C3c more than C3b (Vogel et al., 1984). Although C3b and CVF molecules can form analogous complexes with Bb, the CVF-dependent enzyme differs from the C3b-dependent enzyme. The CVF,Bb enzyme is physicochemically far more stable than C3b,Bb (Vogel and Miiller-Eberhard, 1982; Medicus et al., 1976); it is resistant to inactivation by the regulatory proteins Factors H and I (Lachmann

ALICE H. GRIER and CARL-WILHELM V~CEL

564

and Halbwachs, 1975; Nagaki et al., 1978); it exhibits different kinetic properties (Vogel and MtillerEberhard, 1982; Pangburn and Miiller-Eberhard, 1986); and it does not require C3b for C5 cleavage (Miyama et al., 1975; Von Zabern et al., 1980). These differences in function imply that, despite the structural similarity, some critical difference must exist between C3b and CVF. Another crucial structural difference must exist between CVF and C3c, which cannot form a convertase with Factor B (O’Keefe et al., 1988). One rather substantial difference between CVF and C3 (that so far has not received much attention) is the carbohydrate content of the two molecules which is 7.4% (w/w) for CVF (Vogel and Miiller-Eberhard, 1984) and only 1.7% (w/w) for human C3 (Tack et al., 1979; Tomana et al., 1985). Structural analysis has shown that the alpha and beta chains of human C3 contain one high-mannose-type oligosaccharide chain each (Hirani er al., 1986) in N-glycosidic linkage to asparagine at residues 917 (alpha chain) and 67 (beta chain) (DeBruijn and Fey, 1985). For CVF it has been shown that the alpha and beta chains are glycosylated while the gamma chain is not (Vogel and Miiller-Eberhard, 1984). Based on the carbohydrate composition, it has been suggested that all oligosaccharide chains of CVF are Nglycosidically bonded to asparagine residues (Vogel and Miiller-Eberhard, 1984; Schultz and Vogel, 1987). An initial structural characterization of the major oligosaccharide chain of the molecule revealed a fucosylated symmetric oligosaccharide chain of the complex-type (Bredehorst et al., 1988). The objective of this study was to examine the possible importance of the carbohydrate moieties on CVF to its function. In this paper we describe that the oligosaccharide chains of CVF are required for activation of the alternative pathway of complement by CVF.

MATERIALS

AND METHODS

Materials CVF was isolated from lyophilized cobra venom (N&I nuja siamensis) (Serpentarium Laboratories, Salt Lake City, UT) as described (Vogel and Mi.illerEberhard, 1984). N-glycanase (peptide-N4-(N-acetylb-glucosaminyl) asparagine amidase) (EC 3.5.1.52) (Plummer et al., 1984) was purchased from Genzyme Corporation, Boston, MA. Human serum was obtained by cubital vein puncture of healthy donors. Guinea pig whole complement was obtained from Cordis Laboratories (Miami, FL). Sheep erythrocytes were obtained from Environmental Diagnostics Inc. (Burlington, NC). Guinea pig erythrocytes were obtained by heart puncture of 2/N guinea pigs (NIH, Frederick, MD). Rabbit erythrocytes were obtained by ear vein puncture of New Zealand White rabbits. Rabbit anti-sheep hemolysin was purchased from Colorado Serum Company Laboratories (Denver, CO). Monospecific antisera to human C3 and CVF

were

raised in goats and mice, respectively, as described (Vogel et al., 1984; Grier et al., 1987). Deglycosylation of CVF If not otherwise stated, CVF (approximately 200 pg) was enzymatically deglycosylated by incubation for 6 hr at 30°C with 36 units of N-glycanase in a total volume of 0.5 ml of 0.55 M sodium phosphate buffer at pH 8.6. The extent of deglycosylation was estimated from experiments using ‘251-CVF. After the 6-hr incubation, an aliquot of the reaction mixture containing the N-glycanase treated ‘251-CVF was subjected to SDS-polyacrylamide gel electrophoresis on 9% (w/v) gels under reduction conditions. The Coomassie stained bands of the alpha, deglycosylated alpha, beta, deglycosylated beta and gamma chains were excised from the gels and counted for radioactivity. The bands of untreated or sham treated (incubated without N-glycanase) ‘2SI-CVF electrophoresed on the same gels served as a control. The composition of reaction mixtures containing native, partially deglycosylated and fully deglycosylated CVF molecules was calculated from the experimentally determined percentage of deglycosylated alpha and beta chains using simple probability and assuming independent deglycosylation of the two chains. For example, the probability for native CVF in the reaction mixture containing 70% alpha chains, 30% deglycosylated alpha chains, 70% beta chains and 30% deglycosylated beta chains is 49% (0.7 x 0.7). Similarly, the same reaction mixture is expected to contain 42% (0.7 x 0.3 + 0.3 x 0.7) CVF molecules with one deglycosylated chain (alpha or beta) and 9% (0.3 x 0.3) CVF molecules with deglycosylated alpha and beta chains. PuriJication of deglycosylated CVF Reaction mixtures containing N-glycanase treated CVF (up to 25Opg of protein) were applied to a 1.5 x 0.8 cm concanavalin A-Sepharose column (Pharmacia, Piscataway, NJ), equilibrated with 20mM Tris-HCl, 150mM NaCl, pH 7.5. The breakthrough (peak I) was collected and bound material was eluted with 0.5 M methyl-cc-D-glucopyranoside (Sigma, St. Louis, MO) in the same buffer (peak II) (Grier et al., 1987; Finne and Krusius, 1982). ELISA The ELISA assay for the binding of polyclonal mouse anti-CVF antiserum to CVF and deglycosylated CVF (both native and denatured) was performed as described (Grier et al., 1987) with CVF (1 pg/ml)

plated

at

50 ~l/well

in

microtiter

plates.

Complement consumption by CVF (measured by classical pathway hemolytic activity) The test to determine the ability of CVF or deglycosylated CVF to deplete complement activity in serum was performed by adding different amounts of

Function of carbohydrate CVF or deglycosylated CVF in 100 ~1 of GVB2+ to 100 ~1 of a 1: 2.5 dilution of normal human serum in GVB* + GVB* + is Verona1 buffered saline [VBS; 3.5 mM 5,5-diethylbarbituric acid (Sigma), 143 mM NaCl, pH 7.41 containing 0.15 mM CaCI,, 0.5 mM MgCl, and 0.1% (w/v) gelatin (Sigma) (Vogel and Miiller-Eberhard, 1984). The reaction mixtures were incubated at 37°C. At 30 min intervals, 10 ~1 samples were removed, diluted immediately with 90~1 of ice-cold GVB* + and kept on ice. Subsequently, the remaining complement activity (classical pathway) was determined by a hemolytic assay (Cochrane et al., 1970). To two duplicate 20 ~1 aliquots of the samples, 20 ~1 of hemolysin sensitized sheep erythrocytes (at 5 x 10’ cells/ml in GVB2+ ) were added. The tubes were vortexed and incubated at 37°C for 35min. The reactions were stopped by the addition of 1 ml of cold VBS. Unlyzed cells were pelleted by centrifugation and the absorbance of the supernatants was measured spectrophotometrically at 412 nm. Complement consumption by CVF (measured alternative pathway hemolytic activity)

by

Samples (0.2 p g) of CVF or deglycosylated CVF in 100~1 of GVB*+ were added to 100 ~1 of normal human serum and incubated at 37°C for 3 hr. Subsequently, the samples were placed on ice and the remaining complement activity (alternative pathway) was measured in duplicate 20 ~1 serial dilutions of the samples by adding 20 ~1 of unsensitized rabbit erythrocytes at 5 x 10 cells/ml in GVB2+ The tubes were vortexed and incubated at 37°C for 20min. The reactions were stopped by the addition of 1 ml of cold VBS. Unlyzed cells were pelleted by centrifugation and the absorbance of the supernatants was measured spectrophotometrically at 412 nm. C3 activation

in serum

Determination of C3 cleavage was performed by immunoelectrophoresis using the same serum samples pre-incubated with CVF or deglycosylated CVF, as described above for the consumption of alternative pathway hemolytic complement activity. Immunoelectrophoresis was performed in 1.2% (w/v) agarose in 43.5 mM 5,5_diethylbarbituric acid, pH 8.6, for approximately 2 hr at 5 V/cm at 4°C. Subsequently, the troughs were filled with goat anti-human C3 antiserum and the gels were incubated for 18 hr at 25°C in a humid chamber. Hemolytic

assay for CVF activity

The test for the ability of CVF, deglycoslated CVF, sham treated CVF, or reaction mixtures after Nglycanase treatment (containing native as well as partially and fully deglycosylated CVF) to cause bystander lysis of unsensitized guinea pig erythrocytes was performed by mixing 20 ~1 of normal guinea pig serum, 40 ~1 of the above CVF preparations in GVB2+ and 20 ~1 of guinea pig erythrocytes

565

chains of CVF

at 5 x 10’ cells/ml in GVB’+ (Vogel and MiillerEberhard, 1984). The tubes were vortexed and incubated at 37°C for 40 min. The reaction was stopped by the addition of 1 ml of cold VBS. Unlyzed cells were pelleted by centrifugation and the absorbance of the supernatants was measured spectrophotometrically at 412 nm. Other methods Radiolabeling of CVF was performed by iodination using Na’251 (Amersham, Arlington Heights, IL) and chloramine-T derivatized polystyrene beads (IodoBeads; Pierce, Rockford, IL) (Markwell, 1982). The concn of CVF was determined spectrophotometrically at 280 nm using an extinction coefficient of Et,!: = 0.99 (Vogel and Miiller-Eberhard, 1984) and by the Bradford method (Bradford, 1976). The concn of deglycosylated CVF was initially determined from the radioactivity of a sample generated from ‘251-CVF of known specific radioactivity and by applying the same specific radioactivity for the deglycosylated 12’1CVF. Subsequently, the concn of deglycosylated CVF was determined spectrophotometrically at 280 nm using an extinction coefficient of Ey,$$ = 0.99, and by the Bradford assay using native CVF as standard (compare Results).

RESULTS

Deglycosylation

of CVF with N-glycanuse

To deglycosylate CVF, the native protein was incubated, in the absence of any denaturing agents, with N-glycanase which hydrolyzes the N-acetylglucosamine-asparagine linkages of N-linked ohgosaccharide side chains. The left panel of Fig. I shows the chain structure of CVF (left lane), of the reaction mixture of CVF and N-glycanase after 6 hr at 30°C (center lane), and of the purified N-glycanase treated CVF (right lane) on SDS-polyacrylamide gels under reducing conditions. The Coomassie staining reveals that the treatment of CVF with N-glycanase causes the occurrence of two additional bands with slightly smaller mol. wts than the alpha and beta chains, respectively, but with no change in the gamma chain. The right panel of Fig. 1, showing an identical gel after periodic acid Schiff (PAS) staining, strongly suggests that the reduction in mol. wt of the alpha and beta chains of CVF is due to removal of the oligosaccharide moieties. The left lane confirms the previously reported finding of glycosylated alpha and beta chains with no detectable carbohydrate on the gamma chain of CVF. In the reaction mixture (center lane) the carbohydrate stain only reveals the presence of remaining unaltered alpha and beta chains; the two additional chains present in the reaction mixture as demonstrated by Coomassie stain are not detectable with the carbohydrate stain. Consistent with this observation, the purified N-glycanase treated CVF does not stain with Schiff stain (right lane).

ALICEH. GRER and CARL-WILHELM VOGEL

566

-chain -chain

Y

-chain

PAS Stab

Coomassle Stain

Fig. 1. Chain structure of CVF (left lanes), N-glycanase treated CVF before purification (center lanes), and N-glycanase treated CVF after purification by concanavalin A-Sepharose (right lanes). The Fig. shows identical 9% (w/v) SDS-polyacrylarnide gels under reducing conditions after Coomassie staining

(6 pg per lane) (left panel) and Schiff staining (8 pg pet lane) (right panel). Table 1 shows that approximately 30% of both alpha and beta chains of CVF were deglycosylated by the treatment with N-glycanase. No effect of Nglycanase treatment on the non-glycosylated gamma chain was noted. The experiment described in Table 1 also indicates that N-glycanase treatment does not cause partial proteolytic degradation of CVF, since the total counts recovered from all chains are, within experimental error, the same for N-glycanase treated and sham treated CVF. Increasing the temp of incubation to 37°C or decreasing it to 23°C resulted in lower yields of deglycosylated CVF. An increase in the time of incubation (up to 24 hr), or the addition of the same total amount of N-glycanase in two batches of 18 units each (at 0 and 3 hr), did not significantly increase the degree of deglycosyIation.

Purz$cation of d~g~ycos~i~t~d CVF Deglycosylated CVF was purified from the reaction mixture after N-glycanase treatment by affinity Table

-

1. Extent

._. ... -.--. - _~ Alpha chain Deglycosylated alpha chain Beta chain Deglycosylated beta chain Gamma chain

of deglycosylation

‘%CVF fcpm x 10 ’

I: SE)

12.94 + 0.88 20.84 rt: I .64 2572 _+ 2.20

chromatography on concanavalin A-Sepharose. Figure 2 (top panel) shows a typical elution profile. The breakthrough of the column (peak I) contains the deglycosylated CVF (compare Fig. 1, right lanes). The yield of deglycosylated CVF in the breakthrough was, based on the total counts applied to the column, approximately 5%. Bound material was eluted with methyl-~-D-glucopyranoside (peak II). Analysis by SD~~lyacrylamide gel ele~trophoresis of the bound material revealed the presence of both degiycosylated alpha and beta chains, in addition to the three native chains of CVF (not shown). This result suggests that, after the enzymatic deglycosylation, the reaction mixture contains not only native CVF and completely deglycosylated CVF, but also CVF molecules with one native and one deglycosylated chain, respectively. While deglycosylation of a given chain appears to be complete, deglycosylation of the alpha and beta chains of a CVF molecule seems to occur independently of each other. Assuming this independent deglycosylation of the two chains and applying the observed 30% of

CVF

with N-glycanase~

N-glycanase treated “‘I-CVF (cpm x IO s 2 SE) 8.9610.13 3.92 & 0.05 11.8OrtO.84 5.28 k 0.33 23.64 & 0.72

Deglycosylation W) 30.4 30.9 NA*

Y’z51-CVF was incubated in the absence or the presence of N-glycanase under the conditions described in Materials and Methods. Subsequently, aliquots containing equal amounts of protein (6 fig) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. individual bands were cut from the gels after Coomassie staining and counted for radioaclivity. The data represent mean values from three identical gels run simul~aneousiy. “NA, not applicable.

Function of carbohydrate chains of CVF

30

20

Fraction

Number

Fig. 2. Purification of deglycosylated CVF by concanavalin A-Sepharose affinity chromatography. The top panel shows the elution profile of a reaction mixture containing Nglycanase treated “‘1-CVF. The breakthrough (peak I) contains deglycosyiated CVF. Bound material (peak II) was eluted with 0.5 M methyl-cl-D-glucopyranoside (arrow). The bottom panel shows the elution profile of a reaction mixture containing N-glycanase treated peak II from the first column,

deglycosylation of the two chains (compare Table l), only 9% of the CVF molecules would be expected to be completely deglycosylated, a number that is close to the experimentally obtained yield of 5%. Effect of re~~c~~at~o~of partially deg~~~co~y~ated CVF with N-glycanase The fractions eluted from the concanavalin ASepharose column with methyl-r-D-glucopyranoside *Assuming independent deglycosylation of 30% of both alpha and beta chains of CVF during the first exposure to N-glycanase (compare Table l), the reaction mixture would contain 9% fully deglycosylated molecules, 42% molecules with one glycosylated and one deglycosylated chain (alpha or beta), and 49% native CVF molecules. Peak II from the concanavalin A column (compare Fig. 2, upper panel), which is subject to the second incubation with N-glycanase, therefore consists of remaining native CVF molecules and the partially (one chain) degiycosylated CVF molecules. Assuming again independent deglycosylatio~ of 30% of both alpha and beta chains during the second incubation with N-glycanase, the extent of fully deglycosylated alpha and beta chains would be expected to be 46% each, corresponding to 19% fully deglycosylated CVF molecules, 54% CVF molecules with one giycosylated and one deglycosylated chain (alpha and beta), and 27% native CVF molecules. The observed yields of 52% deglycosylated alpha and beta chains and of 24.6% fully deglycosylated CVF molecules are therefore in good agreement with the expected yields of 46% and 19%. respectively.

567

(peak II; compare Fig. 2, top panel), containing native CVF and partially degIy~osylated CVF (alpha or beta chains), were re-treated with N-glycanase under the same conditions. The samples were subsequently run under reducing conditions on SDSpolyacrylamide gels. The bands of deglycosylated “‘1-CVF were excised and and non-deglycosylated counted, and the percentage of deglycosylation was determined. As shown in Table 2, the second incubation with N-glycanase further increased the extent of deglycosylation of both alpha and beta chains of CVF to approximately 52%. Taking into consideration that a fraction of the CVF molecules in peak II was already partially deglycosylated (alpha or beta chains) prior to the second exposure to the enzyme, the result of 52% final degiycosylation implies that during the second incubation with N-glycanase again approximately 30% of the glycosylated alpha and beta chains were deglycosylated.* Figure 2 (bottom panel) shows the elution profile of the twice N-glycanase treated CVF from the concanavalin A-Sepharose column. The yield of purified completely deglycosylated CVF in the breakthrough (peak I), calculated from the total counts applied, was 24.6%, which is again consistent with the assumption of inde~ndent deglycosylation of the two chains and a 30% degly~osylation of both chains during the second incubation.* Determination

of the concn of deglycosylated CVF

For a comparative analysis of the functional activities of CVF and deglycosylated CVF it was imperative to accurately measure the concn of the deglycosylated CVF. However, it could not necessarily be assumed that the absorption coefficient of the molecule, or the extent of dye binding in the Bradford assay, were unchanged after deglycosylation. We therefore determined the concn of degly~osylated CVF from the radioactivity of a sample generated from ‘**I-CVF of known specific radioactivity and by applying the same specific radio-

Table 2. Extent of second deglycosylanon

of CVF with N-glycanase”

N

-glycanase treated ‘%CVF (cpm x IO-‘& SE)

Alpha chain Deglycosylated alpha chain Beta chain Deglycosylated beta chain Gammachain

6.36 _+0.73 6.89 k 0.41 12.96+0.11 14.03 + 0.1 I 22.99 t 0.29

Deglycosylation (%) 52.2 51.9 NA*

“‘ZSI-CVF was incubated with N-glycanase under non-denaturing conditions as described in Materials and Methods. Subsequently, compieteiy deglycosylated CVF was removed by concanavalin A-Sepharose chromatography. The bound fraction containing native and partially deglycosylated CVF (compare Fig. 2, top panel, peak II) was treated again with N-glycanase under the same conditions. Afterwards, aliquots of the samples (6 Ng) were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Individual bands were cut from the gels after Coomassie staining and counted for radioactivity. The data represent mean values from three identical gels run simultaneously. “NA. not applicable.

ALICEH. GRIERand CARL-WILHELMVOGEL Efict of degIycos~~I~tjon on the agility acthate complement

Reciprocal Dilution of Serum Fig. 3. Effect of deglycosylation on the ability of CVF to consume complement hemolytic activity in human serum, as measured by subsequent alternative pathway activation. The Fig. shows the dilution curves of serum complement hemolytic activity after incubation for 3 hr at 37°C with 0.2 c(g of CVF (0) or 0.2 kg of deglycosylated CVF (m). Human serum incubated without CVF or degiycosylated CVF served as the control (A).

activity for deglycosylated ‘ZsI-CVF. This approach was based on the aSSUUIQiiOnthat enzymatic deglycosylation wilf not change the specific radioactivity (in cpm~mol) of the ‘*%1abe1ed protein. We subsequently determined the absorbance at 280 nm of this sample and the protein concn in the Bradford assay using native CVF as the standard. From the comparison of the concn derived from the specific radioactivity with the concn derived from absorbance and the Bradford assay, respectively, it was learned that the absorption coefficient and the dye binding of deglycosylated CVF are identical to those of native CVF.

of CVF to

Figure 3 shows that incubation of normal human serum with native CVF completely consumes the complement activity, as measured by subsequent alternative pathway activation. In contrast, incubation of normal human serum with deglycosylated CVF has no effect on the hemolytic complement activity. These data demonstrate that deglycosylation of CVF abolishes its ability to consume complement in human serum. Figure 4 shows the time course and dose-response of the consumption of the complement activity in human serum by CVF, as measured by the more sensitive assay for classical pathway activation. Native CVF consumes the hemolytic complement activity in a time- and dose-dependent fashion. Within 120 min of incubation, as little as 0.05 pg of native CVF consumes approximately 50% of the complement activity and 0.5 pg of native CVF completely de-complements the serum. In contrast, no reduction of the complement activity was observed with 0.2pg of deglycosylated CVF. The insert to Fig. 4 shows the dose-response curve of complement consumption by CVF using the 120min time point. The arrow points to the absorbance obtained with 0.2pg of deglycosylated CVF, indicating that the ability to activate complement is reduced by at least two logs upon deglycosylation. Effect qf deglycosylation of CVF induce C3 activation in serum

on its uhility

to

Norma1 human serum was incubated with native or deglycosylated CVF. Subsequently, the extent of C3 activation was estimated from its change of electro-

Time of Incubation (minutes) Fig. 4. Time course and dose-response of complement consumption in human serum by native and deglycosylated CVF, as measured by subsequent classical pathway activation. The Fig. depicts a typical experiment showing time courses of complement consumption by different amounts of CVF (0) and 0.2 pg of deglycosylated CVF (m). The insert shows the dose-response for CVF using the data from the 120 min time point. The arrow indicates the effect of 0.2 pg of deglycosylated CVF.

Function of carbohydrate

.6

Eflect of deglycosyIation CVF

-4 i

4

2

I

0 I

I

0

500

1000

ng

2000

1500 CVF

Fig. 5. Effect of deglycosylation on the ability of CVF to induce complement-mediated lysis of unsensitized guinea pig erythrocytes in guinea pig serum. The Fig. shows the dose-response curves for CVF (0). sham treated CVF (A),

the reaction mixture containing N-glycanase treated CVF before concanavalin A-Sepharose chromatography (0). N-glycanase treated peak II from the concanavalin ASepharose column (i.e. twice treated with N-glycanase but before the second concanavalin A-Sepharose column) (+) and purified

deglycosylated

chains of CVF

CVF (m).

phoretic mobility using immunoelectrophoresis. After incubation of serum with CVF, the C3 had been almost completely activated as indicated by an anodal shift. This shift demonstrates the cleavage of C3 by CVF,Bb to C3b and its subsequent inactivation by factors H and I to iC3b (Lachmann et al., 1982). In contrast, when the serum was incubated with deglycosylated CVF only a low degree of C3 activation occurred, similar to that observed in the control incubation of normal serum with buffer. This low degree of C3 activation upon incubation of serum may be due to the generation of C3(H20) by hydrolysis of the intramolecular thioester of C3, which causes the formation of the short-lived fluid-phase C3 convertase, C3(H,O),Bb (Pangburn et al., 1981).

Dilution

on the hemolytic

activity

of

Figure 5 shows the results of a test for functional activity of CVF, which employs bystander lysis of unsensitized guinea pig erythrocytes. Figure 5 shows the dose-response curves for CVF, sham treated CVF, a reaction mixture of N-glycanase treated CVF before concanavalin A-Sepharose chromatography, N-glycanase treated peak II from the concanavalin A-Sepharose column (i.e. twice treated with Nglycanase but before second purification; compare Fig. 2) and purified deglycosylated CVF. There was no measureable difference in the hemolytic activity of sham treated CVF or CVF. There was also no measurable difference between the hemolytic activity of CVF and the reaction mixture containing Nglycanase treated CVF, presumably due to the small percentage (5%) of fully deglycosylated CVF molecules in the mixture. However, the hemolytic activity of the twice N-glycanase treated sample, which contained approximately 25% fully deglycosylated CVF molecules (see Fig. 2, bottom panel), was reduced by approximately 25%. Most importantly, purified deglycosylated CVF was found to have completely lost its hemolytic activity. Treatment with N-glycanase denaturation of CVF

does not cause gross

We previously reported that CVF can be completely deglycosylated with N-glycanase in the presence of denaturing reagents (Grier et al., 1987). Our subsequent finding of complete loss of the ability of CVF to activate complement after deglycosylation under non-denaturing conditions prompted us to investigate whether the treatment of CVF with Nglycanase causes artificial gross denaturation of the protein unrelated to the removal of the oligosaccharide chains. Figure 6 shows that the binding of a polyclonal mouse anti-CVF antiserum to glyeo-

CVF

log kciprocal

569

Oeglycosylated CVF

of Ab

log Reciprocal Dilution of Ab

Fig. 6. Effect of denaturation on the binding of a polyclonal mouse anti-CVF antiserum to CVF (left panel) and deglycosylated CVF (right panel) by ELISA. The proteins were either native (0) or denatured by boiling for 3 min in the presence of 0.5% (w/v) SDS and 0.1 A4 /?-mercaptoethanol (0).

ALICE H.

570

GRIER and CARL-WILHELM VOGEL

sylated and deglycosylated CVF is reduced by approximately the same extent upon denaturation of the two proteins (approximately 0.75 absorbance units for both proteins at half-maximal binding of the antiserum). With higher antiserum concns the decrease in binding upon denaturation is somewhat greater for deglycosylated CVF, while it is somewhat greater for glycosylated CVF with more diluted antiserum (Fig. 6). This experiment demonstrates that deglycosylated CVF is not grossly denatured and that the extent of its denaturation upon exposure to denaturing agents is not dramatically different from that observed with glycosylated CVF. However, the ELISA method is too coarse to allow any conclusions about the possible presence of conformational epitopes induced by the carbohydrate moieties. Another result apparent from Fig. 6 is that the binding of the polyclonal antiserum to deglycosylated CVF under both native and denaturing conditions is less than to glycosylated CVF under corresponding conditions (both binding curves for deglycosylated CVF are shifted to the left by 1 or 1.5 dilution steps compared to glycosylated CVF). This finding is most likely due to the presence of antibodies in the antiserum directed against oligosaccharide chains of CVF, consistent with our recent identification of monoclonal antibodies to CVF with specificity for its carbohydrate moieties (Grier et al., 1987). Again, this result does not allow any conclusions about the presence of carbohydrate induced conformational epitopes in native CVF. DISCUSSION

The CVF-dependent and C3b-dependent C3jCS convertases differ functionally in several important properties. CVF,Bb exhibits a half-life of 7 hr at 37°C for decay-dissociation into its two subunits (Vogel and Miiller-Eberhard, 1982) compared to the 1.5 min half-life of C3b,Bb (Medicus et al., 1976). C3b,Bb is dissociated by Factor H (Whaley and Ruddy, 1976) and subsequently inactivated by Factors H and I (Pangburn et al., 1977) whereas CVF,Bb is completely resistant to these complement regulatory proteins (Lachmann and Halbwachs, 1975; Nagaki er al., 1978). In order to cleave C5, C3b,Bb requires additional C3b (Daha et al., 1976) whereas CVF,Bb can function as a C5 convertase alone (Miyama et al., 1975; Von Zabern et al., 1980). The two enzymes also exhibit different kinetic properties (Vogel and MiillerEberhard, 1982; Pangburn and Muller-Eberhard. 1986). Another unexplained observation is that, although CVF and C3c are structurally similar (Vogel et al., 1984) some critical structures not present on C3c must exist on CVF which allow CVF to form a convertase with Factor B. An obvious structural difference between CVF and C3 is that the carbohydrate content of CVF (7.4%, w/w) (Vogel and Miiller-Eberhard, 1984) is significantly higher than that of human C3 (1.7%, w/w) (Tomana et al., 1985).

For some glycoproteins it has been found that the carbohydrate portions are essential for the biological function of the molecule, particularly for several glycoproteins in the immune system, including membrane-bound (Remold, 1973; Matthews et al., 1987) and soluble glycoproteins (Nose and Wigzell, 1983; Muchmore et al., 1987a, h). Other studies have shown that the deglycosylation of several glycoproteins, including human CSa-Des-Arg (Chenoweth et al., 1980; Gerard and Hugh, 1981) and granulocyte-macrophage colony stimulating factor (Moonen et al., 1987) significantly increased their biological activity. Since carbohydrates can have crucial functions for the activity of glycoproteins. we investigated the possible contribution of the carbohydrate portion of CVF to its function in the mammalian complement system. In this study we have deglycosylated CVF under non-denaturing conditions with N-glycanase, which hydrolyzes the N-acetyl-glucosaminesparagine linkages of N-linked oligosaccharide chains (Plummer et al., 1984) the only type of carbohydrate-protein linkage presumed to be present in CVF (Vogel and Miiller-Eberhard, 1984; Schultz and Vogel, 1987). While N-glycanase treatment of reduced and denatured CVF resulted in complete deglycosylation (Grier et al., 1987) the removal of carbohydrate from native CVF is much less efficient. Only approximately 30% of both alpha and beta chains of CVF could be deglycosylated during a single exposure to Nglycanase under our experimental conditions. The total number of oligosaccharide chains per CVF molecule is not firmly established yet. Although preliminary evidence suggests that there are three glycosylation sites per CVF molecule (Bredehorst et al., 1988), we did not detect any partially deglycosylated chains. The deglycosylation of the alpha and beta chains seems to proceed independently of each other. The yield of completely deglycosylated CVF (- 5%) was substantially lower than the yield of deglycosylated alpha and beta chains ( - 30%) consistent with our finding that the reaction mixture after N-glycanase treatment contains, in addition to fully glycosylated and fully deglycosylated CVF, CVF molecules with a glycosylated alpha and a deglycoslyated beta chain and vice versa. The comparatively low yield of deglycosylation obtained by the N-glycanase treatment of native CVF compared to denatured CVF is consistent with reports from other investigators describing the action of endoglycosidases. For example, deglycosylation of human C3 by endoglycosidase H required the presence of detergent (Hirani et a/., 1986). The susceptibility of the oligosaccharide chains on native CVF to hydrolysis by N-glycanase may be low due to steric constraints imposed on the enzyme by the native folding of the CVF molecule. An important reason for the low yield of deglycosylation, however, seems to be an inherent property of N-glycanase

Function of carbohydrate itself. While the extent of deglycosylation was dependent on the incubation temp, prolonged incubation (up to 24 hr) at a sub-optimal temp (i.e. 25°C) did not increase the extent of deglycosylation to that observed at 30°C. This observation suggests that the enzyme becomes inactivated during the incubation. This hypothesis is supported by our observation that repeated exposure of CVF to a fresh batch of the enzyme causes further deglycosylation. The initial exposure to the enzyme caused approximately 30% of both alpha and beta chains to be deglycosylated, yielding 5% of completely deglycosylated CVF molecules. The second exposure to fresh N-glycanase yielded 52% deglycosylation of both alpha and beta chains and approximately 25% completely deglycosylated CVF molecules. Taking into consideration that the starting material for the second incubation with N-glycanase (peak II of the concanavalin A column; compare Fig. 2, upper panel) already contained, in addition to fully glycosylated CVF molecules, CVF molecules with one glycosylated and one deglycosylated chain, the observed yield therefore represents approximately 30% indenpendent deglycosylation of the glycosylated alpha and beta chains, identical to the yield during the first exposure to the enzyme (compare footnote on p. 567). Repeated exposure to fresh N-glycanase might therefore represent an experimental strategy to increase the overall yield of deglycosylation. We previously reported that CVF can be completely deglycosylated with N-glycanase under denaturing conditions (Grier et al., 1987). It was therefore imperative to investigate whether the observed complete loss of CVF activity upon deglycosylation might be due to artificial denaturation of the molecule by treatment with N-glycanase and not a consequence of the removal of the oligosaccharide chains. Our strongest experimental support indicating that deglycosylated CVF is not denatured or grossly conformationally changed are the observations that a polyclonal mouse anti-CVF antiserum binds well to deglycosylated CVF and that exposure to denaturing agents reduces the binding by a similar extent to that observed with glycosylated CVF. This result indicates that glycosylated and deglycosylated CVF share many conformational epitopes. Additional, though less strong, evidence suggesting that the deglycosylated CVF is not grossly denatured is our finding that both the extinction coefficient and dye binding were identical for glycosylated and deglycosylated CVF. Another possible reason for the low extent of deglycosylation under non-denaturing conditions may be the presence of a subset of denatured and inactive CVF molecules in the preparation, allowing them to be susceptible to the enzymatic deglycosylation while the native CVF molecules are resistant to N-glycanase. However, several structural data suggest that deglycosylation occurs with native and not with fully or partially denatured CVF mol-

chains of CVF

571

ecules. First, repeated exposure to N-glycanase increases the extent of deglycosylation. This would not be expected if only a finite subset of structurally altered CVF molecules were susceptible to deglycosylation. Second, the deglycosylated CVF shares many epitopes with native CVF, suggesting that the conformation of susceptible CVF molecules cannot be grossly different from those remaining glycosylated. The contention that we are not deglycosylating a denatured or otherwise structurally altered subset of inactive CVF molecules is also supported by functional data. If only an inactive fraction of the CVF molecules were susceptible to deglycosylation by N-glycanase, the overall activity of a reaction mixture should remain constant. However, as shown in Fig. 6, the hemolytic activity of a reaction mixture containing approximately 25% fully deglycosylated CVF molecules was reduced by approximately 25%, indicating that deglycosylation of CVF is associated with a loss of its function. Furthermore, since the same reaction mixture contained in addition approximately 50% of partially deglycosylated CVF molecules, the reduction of the hemolytic activity by only 25% may suggest that complete deglycosylation is required for the loss of CVF activity. The carbohydrate moieties of CVF are importnat for its function since deglycosylation abolishes the ability of the molecule to activate the alternative pathway of complement. Three lines of evidence are presented. First, incubation of human serum with deglycosylated CVF fails to consume the complement activity as measured by subsequent classical and alternative pathway activation. Second, incubation of normal human serum with deglycosylated CVF does not cause C3 activation as demonstrated by immunoelectrophoresis. Third. incubation of guinea pig serum with deglycosylated CVF in the presence of unsensitized guinea pig erythrocytes does not induce bystander lysis of the erythrocytes, indicating a lack of fluid-phase activation of C5 (Pickering et a/., 1969; Miyama et al., 1976; Vogel and Miiller-Eberhard, 1984). Collectively, these results indicate that the removal of the carbohydrate moieties causes the loss of function of CVF. However, our data do not allow the differentiation of whether the carbohydrate structures themselves are required for functional activity, or whether the presence of the carbohydrate structures induces a conformational change in the molecule necessary for its function. The function of the oligosaccharide portion of CVF in molecular terms will require further investigation. Since incubation of serum with deglycosylated CVF does not cause activation of C3 (no change in electrophoretic mobility), C5 (no bystander hemolysis), or Factor B (no consumption of alternative pathway activity), it can be concluded that no functional C3jC5 convertase is formed and that no continuous Factor B activation occurs in serum.

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Deglycosylation of CVF may prevent its binding to Factor B. If this were the case, the carbohydrate moieties are either part of the binding site for Factor B or they maintain a conformation of the CVF molecule necessary for Factor B binding. If binding of deglycosylated CVF to Factor B still occurs, the absence of the carbohydrate side chains may prevent the subsequent activation of Factor B by Factor D. If deglycosylated CVF is still able to form a bimolecular complex with Bb, this complex may exhibit drastically altered enzymatic properties with no apparent C3/C5 convertase activity. Alternatively, deglycosylated CVF may, in the absence of the complement regulatory proteins Factors H and I, be able to form a C3jCS convertase with properties similar, or even identical, to those of the enzyme formed with native CVF. If this were the case, the carbohydrate moieties of CVF would have a protective function against the complement regulatory proteins present in serum. The deglycosylation of CVF may simply expose the binding site for Factor H or a cleavage site for Factor I. It has been described for several other glycoproteins that glycosylation confers a resistance against proteolytic degradation of the molecule (Sharon and Lis, 1980). Human C3 has been shown to contain one highmannose-type oligosaccharide chain each in the alpha and beta chain (Hirani et al., 1986). The glycosylation sites were identified as aparagine residues 917 (alpha chain) and 67 (beta chain) from the cDNA sequence (DeBruijn and Fey, 1985). In contrast, rat C3 has been found to contain one high-mannose-type and two complex-type oligosaccharide chains, all of which are in the alpha chain (Miki et al., 1986). Similarly, two glycosylation sites have been predicted from the cDNA sequence for mouse C3 (asparagine residues 169 and 947) in the alpha chain only (Lundwall et a[., 19846; Wetsel et al., 1984). CVF is glycosylated in its alpha and beta chains (Vogel and Miiller-Eberhard, 1984). Preliminary analysis of its major oligosaccharide chain showed it to be a complex-type oligosaccharide chain (Schultz and Vogel, 1987; Bredehorst et al., 1988). A major difference between the glycosylation of CVF and C3 from human and rat is the total amount of carbohydrate present. CVF has 7.4% (w/w) carbohydrate (Vogel and Miiller-Eberhard, 1984), while human C3 (1.7%, w/w) (Tack et al., 1979; Tomana et al., 1985) and rat C3 (4.0%, w/w) (Miki et al., 1986) have substantially less carbohydrate. Apparently, while all C3 molecules are glycoproteins, the number, position, size and structure of their oligosaccharide moieties vary. These observations may suggest that the carbohydrate portion of C3 molecules may have several biological functions. Except for the binding of human C3 to bovine conglutinin (Hirani et al., 1985), no function of the carbohydrate portion of C3 molecules had been reported. It remains to be determined whether the important role of the carbohydrate portion of CVF for its function in forming an active C3jC5 convert-

ase is unique for CVF or shared by C3b molecules. Our recent observation that two monoclonal antibodies to carbohydrate epitopes of CVF cross-react with carbohydrate epitopes on human C3 (Grier et al., 1987) may suggest that the carbohydrate portion of different C3 molecules share functional relevance.

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