Sheep erythrocytes do not work in the assay of complement in Majorera goat samples in agreement with the results of Venugopal et al.
], who found negligible activity using sheep and cattle erythrocytes, working with goats of undefined breed. This was not observed previously by Olaho-Mukani et al.
] who used sheep RBC with good results with African goats. Oyekan and Barta [13
] showed that the best results for goat complement (breed not stated) were obtained with guinea pig or pig erythrocytes sensitized with goat or cattle antibodies, They also observed lysis with unsensitised rabbit and horse erythrocytes, and low but detectable lysis with sensitised sheep erythrocytes. Noguchi and Bronfenbrenner [28
] did observe that human RBC were usable for testing goat complement. Castro and co-workers [19
] used also Majorera goats for their experiment with rabbit erythrocytes without sensitizing.
The resistance to lysis of sheep RBC may be because sheep complement regulatory proteins (equivalent to CD59 or DAF) are compatible with goat complement proteins. These types of protein protect host cells from autologous complement attack [29
]. These results could be due to a structural similarity between the interaction sites of proteins of both species. For a long time, bovine complement was considered to be nonhemolytic, because it was not able to lyse sheep erythrocytes [30
]. However, in Holstein Bull serum, Chang–Fa et al.
] did not find any relationship between phylogenetic proximity of erythrocyte species to cattle and the degree of hemolysis, attributing this response to the content of the serum of high levels of natural antibodies against RBC of various species. The highest was against guinea pigs and the lowest against cows, sheep, goats and pigs. So, it is possible that in goats something similar happens.
In general, as expected the sensitivity of the assay was improved by sensitization of the erythrocytes with specific antibodies, which enhances the activation of the classical pathway (IgG and IgM) and the alternative pathway (IgG). Buffer was changed because it is difficult to get veronal buffer in some laboratories. Veronal (sodium barbiturate) is the most widely reagent used to measure the activity of the complement system [4
], but other buffers have been used to measure the activity. For example Castro et al.
] used PBS and saline to measure the total complement system activity in kid goat serum although to measure the alternative pathway it was necessary to use the normal EGTA-Mg-gelatin-Veronal buffer. HEPES is a good buffering agent for maintaining physiological pH. The major problem was the storage of RBC in this buffer, which was much poorer than in DGVB++
. For this reason is better to use another buffer, such as Alsever’s solution for storage, to wash the erythrocytes frequently and to minimize as much as possible the storage period in HEPES. The high values for titres in the alternative pathway buffer were unexpected. The “classical pathway” buffer allows activation of all three pathways, while the “alternative pathway” buffer allows only the alternative pathway to work.
It was expected a lower titre for the alternative pathway as the classical pathway usually dominates the activity of the complement system in most species, e.g.
classical pathway activity is detectable in more dilute serum than is lectin or alternative pathway activity [4
]. The same titre value in the two buffer systems would suggest that the activity of the complement system is due to the alternative pathway, but in our work a higher value was observed in the alternative pathway buffer. These results are consistent with previous findings showing that goats have potent alternative pathway activation as was suggested by Venugopal et al.
], and supported by Castro et al.
] and Rodríguez et al.
] who, working with Majorera goat kids, found that the only pathway used was the alternative. The alternative pathway is continually activated at a low controlled rate but amplified by the surface of invading microorganisms [34
]. The environment where the goats live is controlled, e.g.
, nutrition, hygiene or health are supervised by the University staff, and it is thought that microbes that activate the alternative pathway are less pathogenic than those which do not [35
], so that mutation causing loss of the classical pathway (e.g.
loss of C4) might not be a survival factor for these goats.
The higher titre found in the alternative pathway buffer could be due to the higher Mg2+
concentration; Fishelson and Müller-Eberhard [24
], showed that raising the Mg2+
concentration increased the alternative pathway titre. It would be interesting to probe with different of Mg2+
concentrations, Venugopal et al.
] found that higher levels of Mg2+
mM) had an inhibitory effect on the caprine alternative pathway. Matheswaran et al.
], working with buffalo colostrum and with concentrations from 4 to 20
mM of Mg2+
, found no differences, although they chose the concentration of 4
mM as optimum.
The apparent absence of classical pathway could be due to a failure in some component of the classical pathway cascade, possibly a C4 deficiency, or perhaps just because some components were in low concentration, as it occurs in bovine milk with C1q [36
Factor H controls the activity of the alternative pathway C3/C5 convertase by competing with factor B for C3b binding and by serving as a cofactor for factor I, to mediate the cleavage of C3b to iC3b [29
]. Many procedures have been used to purify factor H. In humans, Sim and Discipio [21
] used five chromatography steps to isolate Factor H: first two steps of polyethyleneglycol (PEG) precipitation were used, continuing with L-Lysine- Sepharose 4B chromatography, DEAE-Sephadex A-50 chromatography, Sepharose 6B gel filtration, Hydroxyl-Apatite-Ultrogel chromatography and finally a DEAE-Sepharose CL-6B chromatography. Nakano et al.
] used a shorter procedure to isolate rabbit factor H using various precipitation steps with PEG then three steps of chromatography on DEAE Sephacel; and a further two steps of Sephadex G200 gel filtration. Mhatre and Aston [38
] used a first step of PEG precipitation then three steps of chromatography: DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200 to isolate bovine Factor H. More recently, Factor H has been isolated from porcine seminal plasma by using a Q-Sepharose column then a Matrex Gel Red A column, finishing with an FPLC Superdex 200 column [39
]. The method used in the present study seems to be easier and faster and with good results as shown in Figure . It has three steps: TNP-BSA- Sepharose, protein G Hi-Trap and Superose 6. Binding of human factorH to TNP-BSA had been observed before [24
], but this is the first report where this ligand for affinity purification is used.
C1q has a critical function in host defence and clearance of immune complexes, and for this reason it is desirable to study goat C1q in more depth. Lin et al.
] used 2 different methods for the purification of goat C1q: in one, the procedure was based on two successive precipitation steps at low ionic strength, and followed by an additional purification through a Sepharose CL-6B gel filtration column; and in the other, they used a two-step chromatography, with a BioRex 70 and a Sepharose CL-6B columns. C1q is a protein which has a high affinity for heparin and IgG, so McKay [40
] used these characteristics to make a two-step affinity chromatography procedure to purify C1q from various species (human, rat, rabbit, dog and sheep). Pohl et al.
] isolated C1q using a three-step purification procedure: precipitation from plasma, affinity chromatography with a rabbit IgG-Sepharose column and cleaning up with a rabbit anti-human IgG-Sepharose affinity column. Sasaki et al.
] working with guinea pig serum, combined precipitation with chelating agents, CM-cellulose and Superose 6, and Stemmer and Loos [43
] used a simple and rapid procedure for the purification of C1q from human, guinea pig and mouse serum, with euglobulin precipitation, chromatography on Superose 6B, then Mono S ion exchange.
To establish a simple method to isolate goat complement C1q, previous reports were used as references. As several procedures used immobilised IgG, non-immune human IgG-Sepharose was chosen to begin, followed by a Protein G Hi-trap column to remove contaminant human IgG, ending with ion exchange chromatography on MonoQ and MonoS to remove all the contaminants. The yield was very low, possibly because the concentration of this protein in goats is low, or because human IgG may not be a very high affinity ligand. As noted above, it is possible that C1q is present in low concentration, in that there was very low apparent classical pathway complement activation.
Although little is known about the goat complement genome, only goat C9 and Factor B have been sequenced as far as we know, a goat genome project is in progress [44
] and probably in the next years the sequences of the complement system proteins will be known, so that we will be able to compare the sequences with humans or other species and learn about potential structural or functional differences.
Complement C3 may be the most studied protein of the complement system, due to its abundance and importance in all three pathways. It has been studied and isolated from many species and usually C3 has been isolated in a similar way with a first step of differential precipitation with PEG, continuing with column chromatography (e.g.
Giclas et al.
] for rabbit and Gresham et al.
] for human). Storm et al.
] isolated porcine C3 with PEG precipitation and DEAE-Sephacel chromatography, ending with a size exclusion chromatography with Sepharose CL-6B and a hydroxylapatite chromatography to remove the contaminants. Basta and Hammer [48
], established a two step protocol to isolate C3: PEG precipitation and fast protein liquid chromatography (FPLC) Mono Q ion exchange chromatography, using human and guinea pig. A procedure used in fishes like the spotted wolfish, in which a PEG precipitation, continued by a MonoQ and a Superose 12 exclusion chromatography was described by Abelseth et al.
]. Most recent procedures for the isolation of this protein have been based on the work of Dodds [50
] which was used in the present study and consists of PEG precipitation, and 2–3 ion-exchange steps.
There has been no report of C3 isolation from goats, but in other ruminants, C3 has been purified from cows [51
] using a four step protocol: polyethylene glycol precipitation and chromatography on DEAE-Sephadex A-50, CM-Sephadex A-50 and Sephacryl S-200. In camels, the same procedure was followed by Ouma et al.
]. In the present study the protocol has been simplified to three steps based on a Q Sepharose FF, followed by a MonoQ and a Superose 6.