Characterization and method of isolation for an inhibitor of complement C1

A purified protein, factor J, which has inhibitory properties which prevent the formation or the dissociation of C1 complex and a method of purification for said protein. The method including the following sequential chromatography steps: anion exchange, QAE-Sephadex, heparin-Sepharose affinity, Mono-Q and hydroxylapatite.

The immune system is the power of the body to resist invasion by pathogenic 
organisms, and to overcome such invasion and its ensuing infection, once 
it has taken place. The complement system is important in the immune 
response. Complement is a physiological process which involves many plasma 
proteins that react in a cascading (sequential) effect to mediate a number 
of desirable biologically significant phenomena. Such phenomena include 
modulation of the immune response, facilitation of the transport of immune 
complexes, production of anaphylatoxins which cause release of histamine, 
chemotaxis which is the migration of cells towards the area of complement 
activity, phagocytosis, and lysis of cells. 
The activation of the complement cascade can also cause undesirable 
phenomena, such as inflammation, damage of normal tissue and disease 
states such as the autoimmune diseases. Autoimmune diseases are associated 
with the immune complexes formed against indigenous tissue which are 
associated with the biologically active complement fragments generated by 
the classical portion of the complement cascade. Such diseases include but 
are not limited to: Hashimoto's thyroiditis, systemic lupus erythematosis, 
Goodpasture's syndrome, Graves' disease, myasthenia gravis, insulin 
resistance, autoimmune hemolyic anemia, autoimmune thrombocytopenic 
prupura, and rheumatoid arthritis. 
It is known that the first phase of complement activation begins with Cl. 
Cl is made up of three distinct proteins: a recognition subunit, Clq, and 
the serine proteinase subcomponents, Clr and Cls which are bound together 
in a calcium-dependent tetrameric complex, Clr.sub.2 s.sub.2. An intact Cl 
complex is necessary for physiological activation of Cl to result. 
Activation occurs when the intact Cl complex binds to immunoglobulin 
complexed with antigen. This binding activates Cls which would then react 
with the next plasma protein, C4, to start the cascading effect rolling. 
In terms of the regulation of the complement system, most studies have 
focused on the binding properties of the Cl serine proteinase 
subcomponents, Clr and Cls, for a serum glycoprotein, Cl Inhibitor. 
Another inhibitor that has been identified but whose role in regulating Cl 
function in plasma is not clear is the Clq inhibitor (ClqINH). 
It is important to identify and isolate inhibitors of the complement system 
because by isolating an inhibitor one may be able to control the effects 
of diseases such as those stated above. The inhibitors may provide a basis 
for pharmacologic intervention, either by allowing manipulation of the 
level of an inhibitor, or by providing a model for the chemical synthesis 
of a new inhibitor. 
It is therefore an object of the present invention to provide a method for 
the isolation of an inhibitor of Cl which is functionally and 
antigenically distinct from known inhibitors of Cl. 
It is more specifically an object of the present invention to characterize 
the properties of an inhibitor of Cl, factor J. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, factor J is isolated from body 
fluid, in a multi-column purification procedure. The sequence of columns 
necessary for purification is an anion exchange, QAE-Sephadex, affinity, 
Mono Q,and hydroxylapatite HPLC columns. The purified Factor J has a 
molecular weight (Mr) of about 20,000 daltons, minimal absorption at 280 
nm, and a relatively small number of tyrosine residues. The newly 
discovered protein has been found to inhibit the association of the 
tetrameric complex Clr2s2 with the recognition subunit Clq, and it can 
dissociate the fully assembled-activated Cl complex. 
This inhibitor is functionally and antigenically distinct from other known 
inhibitors of Cl, namely, ClINH and ClqINH. The inhibitory capabilities of 
Cl Inhibitor are the result of its binding to the catalytic subunits of 
Cl, Clr and Cls, and thereby inhibiting Clr and Cls. The Clq Inhibitor can 
only inhibit the assembly of the Cl complex by prior binding to Clq. In 
contradistinction, it has been discovered that factor J does not inhibit 
Cls, and that factor J can both dissociate intact Cl as well as prevent 
its assembly from subcomponents. Thus, factor J is functionally distinct 
from Cl Inhibitor and Clq Inhibitor. 
In its broadest overall aspect, factor J is first isolated and purified and 
then administered in a therapeutic amount to inhibit the undesirable 
activation of the complement cascade.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is based on the discovery of a new, functionally and 
antigenically distinct inhibitor of Cl complex association, factor J. A 
method is described for purifying and characterizing factor J. 
The preparation starts with a sample of body fluid, such as, but not 
limited to, urine and serum, which has been dialyzed. The dialyzate is 
filtered and loaded onto an anion exchange column which has been 
equilibrated with dialysis buffer containing salt. The drop through 
fractions are collected and pooled. 
The pooled fraction is diluted with the starting buffer of the 
QAE-Sepahadex A-50 column and loaded onto the column. Factor J is 
collected in the drop through and early eluted fractions of the linear 
salt gradient. The factor J fractions are pooled and loaded onto a 
heparin-Sepharose affinity column. Factor J elutes between 18 and 20 mS 
during a linear salt gradient when the column is equilibrated at pH 7.4 
and NaCl provides the counter ion. Pools of fractions with factor J are 
concentrated and the buffer exchanged for the starting buffer of the 
Mono-Q column with inhibitors. 
The concentrated solution is loaded on the Mono Q column and the drop 
through fractions are pooled, concentrated and the buffer exchanged with 
phosphate starting buffer of the hydroxylapatite column. The solution is 
then loaded onto a hydroxylapatite column and eluted with an increasing 
linear phosphate gradient. Absorbances at 220, 250 and 280 are measured 
and the final pools are made based on the UV absorbency and inhibitory 
activity. 
The following example is submitted to illustrate but not limit this 
invention. 
EXAMPLE 1 
Human urine was collected from normal donors in 250ml polypropylene bottles 
containing stock amounts of the following inhibitors calculated to achieve 
the following final concentrations: 1 mM phenylmethylsufonyl fluoride 
(PMSF); 5 mM EDTA; 0.01% sodium azide (NaN3); 1 ug/ml leupeptin; 2 mM 
benzamidine-HCl, 1 ug/ml aprotinin. Upon collection of 250 ml of urine the 
bottle was frozen immediately at -70.degree. C. To initiate the 
purification procedure the requisite number of bottles to provide 800-1000 
ml urine were thawed, the urine adjusted to pH 7.4 with a saturated 
solution of Na.sub.2 HPO.sub.4, and dialyzed in 3,500 M.sub.r cut-off 
tubing against 4 changes of 10 liters of 10 mM sodium phosphate, pH 7.4, 2 
mM EDTA, 0.01% NaN.sub.3, 0.5 mM PMSF buffer until the conductivity was 2 
mS, or less. The dialyzed urine was filtered through a 3 um pore 
polypropylene filter, available from Pall-Chisholm Company of Cranston, 
RI, and loaded onto a DEAE-Sephacel, available from Pharmacia LKB 
Biotechnology of Piscataway, N.J., column (5.times.50 cm) equilibrated in 
the dialysis buffer with 0.04 M NaCl added. The drop-through fractions 
were pooled and the pool diluted with 5 volumes of 5 mM Tris buffer, pH 9, 
and applied to a QAE-Sephadex A-50, available from Pharmacia LKB 
Biotechnology of Piscataway, N.J., column (5.times.30 cm) equilibrated in 
1 mM NaCl, 5 mM Tris, pH 9. Factor J activity was about equally present in 
the drop-through fractions and in the early eluted fractions when a linear 
gradient was applied of starting buffer made with 500 mM NaCl. 
Separate pools of factor J were made from the drop-through and eluted 
fractions, and these pools were kept separate over the subsequent 
purification steps, although subsequent studies indicated there was no 
detectable difference in the factor J from the two pools. Each pool was 
loaded on a heparin-Sepharose column (5.times.15 cm) made from crude 
porcine heparin coupled by cyanogen bromide to Sepharose-4B available from 
Pharmacia LKB Biotechnology of Piscataway, N.J., and equilibrated in 25 mM 
NaCl, 50 mM Tris, pH 7.4. Factor J activity eluted between 18-20 mS during 
a linear gradient of starting buffer made with 1.5 M NaCl. Pools of 
fractions with factor J activity were concentrated and buffer exchanged by 
ultrafiltration using a cellulose 1000 M.sub.r cut-off membrane, 
Spectra/Por type C, available from Spectrum Medical Industries of Los 
Angeles, CA, into the starting buffer for the Mono Q, 40 mM NaCl, 10 mM 
sodium phosphate, pH 7.8, 2 mM EDTA, 0.01% NaN.sub.3, 5 mM PMSF. The 
concentrated pools were loaded onto a Mono Q HPLC column, HR 5/5, 
available from Pharmacia LKB Biotechnology of Piscataway, N.J., and the 
drop-through fractions pooled, concentrated, and buffer exchanged into the 
starting buffer for hydroxylapatite, 10 mM sodium phosphate, pH 7.4, 0.01 
mM CaCl.sub.2 as described above. The concentrated pools were loaded onto 
a HPHT hydroxylapatite HPLC column available from Bio-Rad of Richmond, CA, 
and eluted with a linear gradient of 10-400 mM sodium phosphate pH 7.4, 10 
um CaCl.sub.2. As seen in FIG. 1, absorbances at 220nm, 30, 254nm 31, and 
280 nm 32 were measured simultaneously using a diode-array 
spectrophotometer, Hewlett-Packard #1040A, available from Hewlett-Packard 
Analytical Instruments of Avondale, PA. Final pools were made based on UV 
absorbency, 30, 31, 32, and inhibitory activity, 33. 
The following characterization data represents specific results of factor J 
purified according to example 1. The factor J isolated is a protein with 
the following properties. FIG. 2 is an autoradiograph of unreduced, Lane 
42 and reduced, Lane 43, .sup.125 I-factor J, run on a 3-20% slab SDS-PAGE 
gel. The major bands of factor J had a mobility of 18,400 M.sub.r, 44, 
which did not change with reduction 45. Repeated analysis of factor J 
revealed a molecular weight which varied from 18,000, 44, to 22,000, 45. 
This variation is inherent in this method. A second prominent band was at 
200,000 M.sub.r, 46. Molecular weight determination was based on the 
.sup.14 C labeled protein standards: myosin (200,000), phosphorylase b 
(92,500), bovine serum albumin (69,000) ovalbumin (46,000) carbonic 
anhydrase (30,000) and lysozyme (14,100). We believe the true molecular 
weight to be about 20,000 M.sub.r because manipulations such as storage, 
heating, exposure to low pH or reducing agents increased the relative 
amounts of the 200,000 M.sub.r and decreased the relative amount of the 
20,000 M.sub.r form. 
Isolated factor J has the capacity to agglutinate the erythrocytes of 
various species (human, rabbit, guinea pig and sheep erythrocytes have 
been tested, and all are positive). This agglutination becomes apparent 
after the factor J has passed through QAE-Sephadex. The agglutination 
titer and functional inhibitory titers are roughly parallel. The 
agglutination can be inhibited by commercial heparin. 
The amino acid composition of isolated human urine factor J revealed a 
relatively small amount of tyrosine, about 8.7 residues per 1000, which is 
consistent with the poor reactivity of factor J in Folin Assays. In 
addition, UV spectra of purified factor J, FIG. 3, suggests a low 
tryptophan value which is demonstrated by the minimal absorption of 
purified factor J at 280nm, 55. 
Results indicate that factor J is not an enzyme. Factor J inhibition occurs 
rapidly as can be seen in FIG. 4. Factor J reached maximum inhibitory 
potential within approximately five minutes, 61. Inhibitory potential was 
measured using a functional hemolytic assay. FIG. 5 shows that factor J 
activity (% inhibition) is not affected by temperature. There was no 
significant change in activity at temperatures ranging from 4.degree. C., 
62, to 37.degree. C., 65. FIG. 6 shows a reciprocal plot of the data which 
indicates that factor J inhibition is noncompetitive, 68. This suggests 
that the catalytic subunit and factor J are binding reversibly, randomly 
and independently at different sites. Accordingly factor J could be 
binding to Clq directly or it could be binding to Clq once its is bound to 
Clr.sub.2 s.sub.2. 
Factor J did inhibit association of the Cl complex as measured by factor 
J's ability to inhibit the precipitation of .sup.125 I-Clq in the presence 
of Clr and Cls, FIG. 7. This titration profile, 71, was very similar to 
that obtained when the dose response of factor J inhibition of Cl 
formation in the hemolytic assay, 72. Both assays were measured over the 
same concentration range of polypeptide. The difference in the shape of 
the inhibition curves for .sup.125 I-Clq interaction with Clr.sub.2 
s.sub.2, 71, and the inhibition of Cl hemolytic activity, 72, emphasizes 
that factor J inhibits the Clq and Clr.sub.2 s.sub.2 reaction in a 
saturable manner consistent with direct binding to Cl, whereas the 
inhibition of Cl hemolytic function follows a sigmoidal 72 response 
consistent with the complex kinetics of erythrocyte lysis induced by 
diluted serum. 
Although factor J has been shown to inhibit Clq association with Clr.sub.2 
s.sub.2, the mechanisms for this inhibition are not the same as that of Cl 
inhibitor or Clq inhibitor. Cl inhibitor acts by binding to both catalytic 
subunits of Cl, Clr and Cls. An assay measuring esterase activity of 
purified Cls, FIG. 8 compares factor J inhibition for Cls, 82, with 
inhibition of the Cl inhibitor for Cls, 83. As seen in FIG. 8, Cls in the 
presence of factor J, 82, or buffer alone, 81, show comparable amounts of 
esterase activity, whereas, the addition of Cl Inhibitor resulted in a 
significant decrease in Cl esterase activity, 83. Clq inhibitor acts by 
binding to Clq and thereby preventing the catalytic subunits from binding 
to Clq. FIG. 9 demonstrates that factor J does not bind to Clq under 
conditions in which the Clq Inhibitor could bind Clq. Partially purified 
Clq inhibitor bound to and precipitated .sup.125 I-Clq, 101, whereas, 
purified factor J did not bind to .sup.125 I-Clq in the fluid phase to 
permit precipitation of the .sup.125 I-Clq, 102. 
Factor J can also inhibit the human alternative complement pathway in an 
assay utlizing sheep erythrocytes bearing human C3b, and purified factor 
D, factor B, and peperidin. The process step in the alternative pathway 
where factor J inhibits is not yet known. 
Antigenic results indicate that factor J is present in human serum and it 
does not cross react with antigen for Cl Inhibitor. FIG. 10 shows that 
goat anti-human serum precipitates radiolabeled factor J, 121, above the 
background level precipitated by normal goat serum, 122. Goat anti-Clq, 
123, did not cause any precipitation of radiolabeled factor J above 
background. In addition, FIG. 11 demonstrates that factor J is not 
antigenically related to Cl Inhibitor. The anti-Cl Inhibitor did not 
specifically absorb the factor J, 111, as compared with anti-5, a control, 
112, whereas the anti-Cl Inhibitor was able to specifically absorb 
.sup.125 I-Cl Inhibitor under the same conditions, 113. 
Having above indicated a preferred embodiment of the present invention it 
will occur to those skilled in the art that modifications and alternatives 
can be practiced within the spirit and scope of the invention. It is 
accordingly intended to define the scope of the invention only as 
indicated in the following claims. 
EXAMPLE 2 
Approximately 100-200 ml of serum is collected and saturated to 15% 
(weight/weight) with polyethylene glycol available from Sigma Chemical Co 
of St. Louis, MO. The saturated solution is kept at 4.degree. C. for 30 
minutes and then centrifuged. The precipitate is collected and the 
supernatant is discarded. 
The precipitate is solubilized with pH 7.5 NaCl phosphate buffer and 
further diluted with water to adjust the solution to a conductivity of 
about 4 mS. This adjusted solution is loaded onto a DEAE-Sephacel, 
available from Pharmacia LKB Biotechnology of Piscataway, N.J., column 
(5.times.50 cm). The non-absorbed material is collected, pooled and 
adjusted to pH 9. The adjusted material is then applied to a QAE-Sephadex 
A-50, available from Pharmacia LKB Biotechnology of Piscataway, N.J., 
column (5.times.30 cm) equilibrated in 1 mM NaCl, 5 mM Tris, pH 9. The 
effluent is collected, pooled and adjusted to pH 7.2. 
The adjusted effluent pool was loaded on a heparin-Sepharose column 
(5.times.15 cm) made from crude porcine heparin coupled by cyanogen 
bromide to Sepharose-4B, available from Pharmacia LKB Biotechnology of 
Piscataway, N.J., and equilibrated in 25 mM NaCl, 50mM Tris, pH 7.4. 
Factor J activity eluted between 18-20 mS during a linear gradient of 
starting buffer made with 1.5 M NaCl. Pools of fractions with factor J 
activity were concentrated and buffer exchanged by ultrafiltration using a 
cellulose 100 M.sub.r cut-off membrane, Spectra/Por type C, available from 
Spectrum Medical Industries of Los Angeles, CA, into the starting buffer 
for the Mono Q column, 40 mM NaCl, 10 mM sodium phosphate, pH 7.8, 2 mM 
EDTA, 0.01% NaN.sub.3, 5 mM PMSF. The concentrated pools were loaded onto 
a Mono Q HPLC column, HR 5/5, available from Pharmacia LKB Biotechnolgy of 
Piscataway, N.J., and the drop-through fractions pooled, concentrated, and 
buffer exchanged into the starting buffer for the hydroxylapatite column, 
10 mM sodium phosphate, pH 7.4, 0.01 mM CaCl.sub.2. The concentrated pools 
were loaded onto a HPHT hydroxylapatite HPLC column available from Bio-Rad 
of Richmond, CA, and eluted with a linear gradient of 10-400 mM sodium 
phosphate pH 7.4, 10 uM CaCl.sub.2. Pools were made based on UV absorbency 
and inhibitory activity. This pool is then applied to a Vydac C4 reverse 
phase column, available from Vydac of Hisperia, CA, which has been 
equilibrated with 0.1% trifluoracetic acid (TFA) in water. The factor J is 
eluted in a gradient made with the equilibration buffer and 95% 
acetonitrile in 5% water, 0.1% TFA. The elution rate is 1ml/min and the 
factor J peak elutes at about 19 min in a 30 minute run. The fractions are 
dried down and reconstituted in 0.1 M NH.sub.4 HCO.sub.3 buffer for 
assaying. 
In practice, factor J is isolated, purified and administered in a 
therapeutic amount to inhibit the undesirable activation of the complement 
cascade system.