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Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879
ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(5): 483-488
CN 31-1300/Q |
A
Highly Active Anticomplement Factor from the Venom of Naja kaouthia
( Kunming Institute of Zoology, the
Chinese Academy of Sciences, Kunming 650223, China )
CVF
binds to factor B in serum to form the complex CVF.B, which is cleaved by
factor D into CVF.Bb and Ba. The CVF.Bb is a C3/C5 convertase. This C3/C5
convertase triggers the alternative complement pathway, and results in
depleting of complement. For this reason, CVF is often used in immunological
research as a tool for decomplement in vitro and in vivo[10–13].
Recently, CVF showed clinical potential in overcoming hyperacute rejection in
xenotransplantation[14,15]. That prompted us to isolate anticomplement
factor from cobra venom.
In
this paper, isolation, biochemical properties and biological activity of a
highly active anticomplement factor from the venom of Naja kaouthia are
presented.
1.1 Materials
The
lyophilized Naja kaouthia crude venom from South Yunnan, China was
obtained from Kunming Institute of Zoology, the Chinese Academy of Sciences,
Kunming, China. Hartley guinea pigs (200-300
g) were provided by Department of Laboratory Animal, Kunming Medical College,
Kunming, China. Wistar rats (200-300
g) were provided by Laboratory Animal Center, Tongji Medical University, Wuhan,
China. Rhesus monkeys (4-5
kg) were provided by Laboratory Animal Center, Kunming Institute of Zoology,
the Chinese Academy of Sciences, Kunming, China. SP-Sephadex-C-25, Q Sepharose
HP and Sephadex G-150 were purchased from Amersham Pharmacia Biotech. ÄKTA
explorer is product of Amersham Pharmacia Biotech. 476A protein sequencer is
product of Applied Biosystem Inc. Low molecular marker, pI marker and precast
gel used in electrophoresis came from Amersham Pharmacia Biotech. High
molecular marker was obtained from Shanghai Institute of Biochemistry, the
Chinese Academy of Sciences, Shanghai, China. N-acetylneuraminic acid is
product of Sigma. Rabbit anti-sheep red blood cell serum was obtained from
Shanghai Institute of Biological Products, Ministry of Health, China. Reagent
for protein quantitation is product of Bio-Rad. All other chemicals are
commercial preparations of analytical grade.
1.2 Isolation
3.0
g of crude venom from Naja kaouthia was dissolved in 20 ml sodium
acetate buffer (0.05 mol/L pH 6.0). The solution was applied to a
SP-Sephadex-C-25 column (2.6 cm×50
cm) equilibrated with the same buffer. Fractions with complement-depleting
activity were pooled and dialyzed against sodium phosphate buffer (0.01 mol/L,
pH 7.4). The material was applied to a Q Sepharose HP column (1.6 cm×20
cm) on ÄKTA explorer. After the column was thoroughly washed, protein was
eluted with a linear NaCl gradient (0 to 0.6 mol/L) at a flow rate of 300 ml/h.
Fractions containing anticomplement activity were pooled, concentrated and
dialyzed against sodium phosphate buffer (0.01 mol/L, pH 7.4). After
precipitate was removed by centrifugation, the supernatant was applied to a
Sephadex G-150 column (2.6 cm×90
cm) equilibrated with 0.01 mol/L sodium phosphate buffer containing 0.1 mol/L
NaCl, pH 7.4. The column was eluted with the same buffer at a flow rate of 12
ml/h. Fractions containing anticomplement activity were pooled and stored at
-20 ℃.
1.3 Electrophoresis
SDS
polyacrylamide gel electrophoresis was performed according to Laemmli[16]
using 7% polyacrylamide under non-reducing conditions and 12% polyacrylamide
under reducing conditions. Molecular weight under reducing conditions was
determined using a method for glycoprotein[17]. Protein was stained
with Coomassie brilliant blue. Electrophoresis in absence of SDS was performed
with 7% polyacrylamide gel according to the method of Laemmli[16].
Pharmacia
PhastSystem was employed to determine isoelectric point according to the method
recommened by Amersham Pharmacia Biotech.
1.4 Carbohydrate
analysis
Periodate-Schiff
reagent was used to stain carbohydrate according to the method of Guan et al[18].
Neutral sugar was determined according to the method of Zhang[19].
Sialic acid was determined according to the method of Guan et al[18].
1.5 Amino
acid analysis
Amino
acid analysis was performed on a Hitachi automatic amino acid analyzer, model
835, according to Spackman et al[20].
1.6 N-terminal
sequencing
Edman
degradation method was employed to determine the N-terminal sequence by a 476A
protein sequencer from Applied Biosystem Inc.
1.7 Complement
inactivation in vitro and in vivo
1.7.1 Assay
for anticomplement activity Anticomplement
activity of the anticomplement factor was measured according to the method of
Takahashi et al[3]. 0.1 ml of each sample was preincubated
with 0.5 ml guinea pig serum diluted (1∶100)
with glucose-gelatin veronal buffer for 20 min at 37 ℃.
0.4 ml of sheep red blood cells (5 ×108
cells/ml) in glucose-gelatin veronal buffer which had been sensitized by
anti-sheep red blood cell serum were added and incubated for a further 15 min
at 37 ℃
with occasional shaking. After that, 2 ml of cold phosphate-buffered saline
were added to each tube and centrifuged for 10 min at 1 800 r/min. The optical
density of supernatant was measured at 541 nm on a Pharmacia Biochrom 4060
spectrophotometer. Fractions containing anticomplement activity were pooled and
concentrated.
1.7.2 Determination
of anticomplement activity unit 0.1
ml of 2-fold serial dilutions in glucose-gelatin veronal buffer were tested for
inhibition of lysis as mentioned above. The unit of anticomplement activity was
defined according to Ballow et al[1] as the quantity of
anticomplement factor in 0.1 ml which caused 50% inhibition of lysis in the
above assay.
1.7.3 Complement
inactivation in vivo Three
Wistar rats and three Rhesus monkeys were gaven a single injection (i.v.) of
0.02 mg/kg of the anticomplement factor. The rats were bled after 0.25, 0.5, 1,
2, 3, 4, 5, 6, 8 days by tail and the monkeys were bled after 1 day. Their sera
were assayed for remaining complement activity according to Ballow et al[1]
and Kabat et al[21], respectively.
1.8 Protein quantitation
Protein
concentration was determined by Coomassie brilliant blue method recommended by
Bio-Rad.
2.1 Purification and biochemical
properties of Naja kaouthia anticomplement factor
3.0
g of crude venom dissolved in sodium acetate buffer (0.05 mol/L, pH 6.0) was
applied to a column of SP-Sephadex-C-25. The complement-depleting activity was
found in break-through fractions (Figure not shown), which were pooled and
applied to a column of Q Sepharose HP in sodium phosphate buffer (0.01 mol/L,
pH 7.4) on ÄKTA explorer. As shown in Fig.1, the last peak showed
anticomplement activity. The fractions containing anticomplement activity were
concentrated and applied on a Sephadex G-150 column equilibrated with 0.01
mol/L sodium phosphate buffer containing 0.1 mol/L NaCl, pH 7.4. The
anticomplement activity was found in the second peak (Fig.2). The yield of this
fraction, isolated from 3.0 g of the crude venom of Naja kaouthia, was
approximately 8 mg.
Fig.1 Q Sepharose HP chromatography of
the void peak from SP-Sephadex-C-25
The
last peak showed anticomplement activity (indicated by the hatched area).
Protein concentration (absorbance at 280 nm, --), conductivity (-------) are indicated.
Fig.2 Sephadex G-150 gel filtration of
the fractions with anticomplement activity from Q Sepharose HP
The
second peak contained anticomplement activity (indicated by the hatched area).
Fig.3 Polyacrylamide gel
electrophoresis of purified anticomplement factor
(A) PAGE under non-reducing conditions in
absence of SDS; (B) SDS-PAGE under non-reducing conditions; (C) SDS-PAGE under
reducing conditions. 1, purified Naja kaouthia anticomplement factor; 2,
high molecular marker; 3, low molecular marker.
The
N-terminal sequences of the three polypeptide chains are shown in Fig.4.
N-terminal sequences of the two minor bands (g-chain) are homogenous.
Fig.4 The N-terminal sequences of the
three polypeptide chains of the anticomplement factor from the venom of Naja
kaouthia
All
polypeptide chains could be stained by periodate-Schiff reagent(Figure not shown).
That indicated the anticopmlement factor was a glycoprotein. Neutral sugar was
determined to be about 1.78%, and sialic acid was determined to be about 0.38%.
The
amino acid composition assay showed that the anticopmlement factor contained
37.7% neutral, 26.8% acidic, 24.5% polar and 11.1% basic amino acids.
Comparison of amino acid compositions of various CVF was listed in Table 1.
2.2 Anticomplement
activity of Naja kaouthia anticomplement factor
The
purified anticomplement factor was assayed for anticomplement activity and
found to possess 1 unit of anticomplement activity per 0.66 mg protein. Its
anticomplement activity is much higher than other CVF reported[1, 3, 5,
22]. Comparison of anticomple-ment activity of various CVF was listed in
Table 2.
Fig.5
showed the hemolytic activity of rat serum after a single injection (0.02 mg/kg,
i.v.) of purified anticomplement factor. After 6 h the activity was less than
3.5% and reached normal levels after 6 days. After a single injection (0.02
mg/kg, i.v.) of purified anticomplement factor, the hemolytic activity of
monkey serum was less than 1% within 24 h(Figure not shown). No morbidity or
mortality was observed. This Naja kaouthia anticomplement factor showed
higher activity in vivo than other CVF reported[14, 15, 23, 24].
Comparion of complement-depleting activity in vivo of various CVF was
listed in Table 3.
Fig.5 Serum complement
activity in rats after a single injection (0.02 mg/kg, i.v.) of purified
anticomplement factor(means of 3 animals)
3 Discussion
The
anticomplement activity of cobra venom has been known since the early 1900's[1].
Various anticomplement factors have been isolated fromthe venoms of Naja
naja[1], Naja naja siamensis[2], Naja
naja atra[3, 5] and Naja naja kaouthia[4].
The
anticomplement factor we purified from the venom of Naja kaouthia was
homogeneous on polyacrylamide gel electrophoresis. It appeared to be a protein
with a molecular weight of about 149 kD by SDS-polyacrylamide gel
electrophoresis under non-reducing conditions. And under reducing conditions,
two major bands (a-chain, b-chain) and two minor bands (g-chain) appeared.
These results are in agreement with the results of Pepys et al[22],
Eggertsen et al[2] and Vogel et al[4].
Vogel et al[4] reported that g-chain even exhibited five
bands in some gels. In our gels g-chain displayed two bands clearly. N-terminal
sequence of the two minor bands was found to be homogenous. The
microheterogeneity of g-chain might be caused by carbohydrate moieties or small
differences in the carboxyterminal sequence. All bands could be stained by
periodate-Schiff reagent. The results presented here suggest that the
anticomplement factor is a glycoprotein consisting of three polypeptide chains
linked together by disulfide bonds.
CVF
from Naja naja siamensis[2] and Naja naja atra[3,
5] has been reported to contain carbohydrate in all three chains. Vogel et
al[4] reported that no carbohydrate was found in the g-chain of
the Naja naja kaouthia anticomplement factor. In contrast, our result
showed that all three chains of Naja kaouthia anticomplement factor
contained carbohydrate.
The
amino acid composition of the purified anticomplement factor was similar to
those isolated from Naja naja siamensis[2], Naja naja atra[3],
and Naja naja kaouthia[4]. A relatively high proportion of
acidic amino acid in the anticomplement factor explained its pI value.
This
anticomplement factor showed much higher anticomplement activity in vitro
and in vivo than other anticomplement factor reported. One unit of
anticomplement activity is 0.66 mg protein, compared with 4-5
mg[1], 4 mg[3], 12.72 mg[5] and 1-2
mg[22]. When a single dose of 0.02 mg/kg of this anticomplement
factor was administered to rats and monkeys, the hemolytic activity was less
than 1% within 12 h and 24 h, respectively. Other CVF, to achieve the same
target, 60 u/kg (i.v.)[23], 80 u/kg (i.v.)[24] to rat,
0.5 mg/kg(i.m.)＋0.25
mg/kg (i.v.)[15], 60 u/kg (i.v.)[14] to baboon would be
needed. The reason for higher anticomplement activity of this protein than
others is obscure at present, and further investigation is needed.
Successful
allotransplantation has resulted in frequent donor organ shortages.
Xenotransplantation between different species such as pig to human may
potentially resolve this problem. The most formidable barrier to
xenotransplantation is hyperacute rejection. Complement plays a key role in
this rejection. Depleting complement can overcome hyperacute rejection efficiently
and prolong xenograft survival remarkably[14, 15, 23, 24]. Our
results showed that safe and effective depletion of complement could be
achieved with this highly active anticopmlement factor. This highly active
anticomplement factor may not only be used in investigating the role of
complement in host defense or pathogenesis of disease, but most of all, it
shows potential in xenotransplantation research and application.
Acknowledgements
The authors are indebted to Dr. SUN Zhong-Sheng(Medical School, Cornell
University) for revising the manuscript, to Dr. JIANG Yian-Jun, Dr. JIN Yang,
Dr. LAI Ren and Dr. WEI Qin, to Mr. LI Dong-Sheng and Mr. ZHU Shao-Wen for
their technical assistance.
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Received: April 23, 2001 Accepted: May
29, 2001
This work was supported by a grant from
the Key Research Programs of Yunnan Province, China (No.C0007Z)
*Corresponding author: Tel,
86-871-5192476; Fax, 86-871-5191823; e-mail, [email protected]