Structure and evolution of artiodactyla haptoglobins

Comp. Biochem. Physiol., Vol. 60B~ pp. 389 to 396. © Pergamon Press Ltd 1978. Printed in Great Britain

0305-0491/78/0715-0389502.00/0

STRUCTURE AND EVOLUTION OF ARTIODACTYLA HAPTOGLOBINS WALKER H. BUSBY JR and JAMES C. TRAVIS The Neurobiology Program, Medical Research Wing 206-H, The University of North Carolina at Chapel Hill, Chapel Hill, N.C. 27514, U.S.A.; and Biology Department, The University of North Carolina at Charlotte, Charlotte, N.C. 28213, U.S.A. (Received 19 September 1977)

Abstract--1. Artiodactyla haptoglobins (Hps), goat, sheep and cattle (family Bovidae), and pig (family Suidae) were structurally characterized. 2. The polymeric Hp systems of goat, sheep and cattle were similar to the polymeric human Hp system, while the monomeric system of pig was more comparable to the monomeric human form. 3. All members of the Artiodactyla (family Bovidae) examined exhibited a large polypeptide subunit, comparable to that of the fl subunit of human Hp. 4. In addition, a small subunit, similar in molecular weight to the human ct2 subunit, was demonstrated. Pig Hp was shown to have two subunits, one slightly larger than the human fl subunit and the other intermediate in size to the human ct1 and ct2 subunits. 5. Immunoelectrophoretic and immunodiffusion studies indicated complete cross reactivity among the polymeric Artiodactyla Hps. 6. The polymeric Hps do not, however, cross react with the monomeric pig Hp.

INTRODUCTION

A hemoglobin (Hb) binding protein, haptoglobin (Hp), was discovered in human plasma by Polonovski & Jayle in 1938. This protein, an ~2-globulin, binds the globin moiety of Hb (Laurell & Gronvall, 1962) in a stoichiometric ratio (Jayle et al., 1956). To a great extent, the concentration of Hp in the plasma determines the renal threshold of Hb in the plasma, as the formation of the large H p - H b complexes precludes glomerular filtration (Laurell & Nyman, 1957; Bunn et al., 1967). The rise of the ~:-globulins in association with many disease states is primarily due to Hp (Jayle & Boussier, 1954). Tissue damage or inflammation results in high Hp levels, especially evidenced in connective tissue disorders such as rheumatic fever and rheumatoid arthritis (Nettlebladt & Sundblad, 1967; Noonan et al., 1968). Hp is also considered as an acute phase protein associated with infections (Moretti, 1968). Hp is a glycoprotein which is composed of four polypeptide chains, two c¢ and two fl subunits, linked by disulfide bridges (Smithies et al., 1962). From gel electrophoretic studies, humans have been shown to exhibit three phenotypic patterns (Smithies & Walker, 1956). The sieving effect of polyacrylamide gels results in either a single band, Hp 1 (monomerism), or multiple bands, H p 2-1 or Hp 2 (polymerism). This polymorphism is due to the presence of two alleles at the Hp ~t locus, Hp c¢~ and Hp ~2, encoding for the ct~ and c¢2 polypeptide chains (Connell et al., 1962). Hp 1 contains only c¢~ chains, Hp 2-1 both c¢1 and ~2, and Hp 2 only ct2 (Smithies et al., 1962; Smithies et al., 1966). Identical fl polypeptide chains are common to all three phenotypes (Cleve et al., 1967). It thus appears that the c¢2 polypeptide chain initiates polymerization, resulting in a series of Hp molecules of increasing molecular weight.

Structurally the ~2 polypeptide chain is similar to the al chain but nearly twice as long, the molecular weights being 17,000 and 8900, respectively (Smithies et al., 1962; Connell et al., 1966). It was felt, therefore, that the Hp a2 allele arose from a partial gene duplication of the Hp al allele (Smithies et al., 1962). Sequence studies have upheld this idea (Black & Dixon, 1968). The fl polypeptide chain, containing the entire Hp carbohydrate (CHO) moiety, has a molecular weight of 40,000 (Chattel & Moretti, 1966). The CHO content is estimated to be 18.6% of the native glycoprotein (Cheftel & Moretti, 1966). As recently as 1970, all non-human mammals, including primates that had been tested for Hp, exhibited a monomeric form similar to human Hp 1. However, during the early 1970s some members of the Artiodactyla (family Bovidae), including goats (Travis et al., 1970), sheep (Jarrett, 1972; Travis & Sanders, 1972), and cattle (Goodger, 1972) were found, by gel electrophoresis, to exhibit polymeric Hp's. Pig Hp (family Suidae), however, was demonstrated to have only a monomeric form (Graetzer et al., 1965). Therefore, it was postulated that a Hp ct2-1ike allele exists in Artiodactyla of family Bovidae, while in family Suidae no ~2 allele is present (Travis et al., 1975). Since human and Bovidae are relatively distant on an evolutionary scale, these investigators further postulated that similar but independent genetic events, i.e. partial duplication of a Hp ~1 precursor allele, occurred in both evolutionary lines, and that this genetic event occurred after divergence of the families Bovidae and Suidae. Human Hp has been characterized to a great extent, including molecular weight determinations and amino acid analysis (Smithies et al., 1962; Smithies et al., 1966; Fuller et al., 1973; Barnett et al., 1972). Characterization of the Artiodactyla Hp's, especially that of goat, has been initiated (Travis et al., 1975). Goat Hp was demonstrated to exhibit two 389

390

WALKER H. BUSBY JR and JAMES C. TRAVIS

subunits, one of 40,000 molecular weight (//-like), and a second of 18,500 molecular weight (~2-1ike). Therefore, the Artiodactyla and human Hp systems appear to exhibit some structural similarity. In this report further data are presented elucidating the structure of the Artiodactyla Hp's.

ried out for 5-6 hr, 130 V, 120 mA. Gels were stained overnight in 0.1°~ Coomassie Blue in 10~,,, glacial acetic acid. Destaining was accomplished with 10°o glacial acetic acid. Carbohydrate determinations were made according to the phenol-sulfuric acid test (Colowick & Kaplan, 1966). Optical density was determincd at 485 nm on a Beckman 25 UV spectrophotometer. In order to examine immunological relatedness, Artiodactyla Hp's were subjected to immunodiffusion and immunoelectrophoretic assays. Goat, sheep, cow and pig anti-Hp sera were prepared in the following manner: 1 ml solutions (10mg protein in 2ml of 0.9~;, saline + 2ml adjuvant) were injected subcutaneously into rabbits. Approximately 30 days later, 0.25 ml solutions (5 ~ng protein/ml salinet were injected intravenously. The intravenous injection was repeated after 2 days. 5 days after this, rabbits were bled by cardiac puncture. 10°,g EDTA (0.20ml/10ml of blood) was used as an anticoagulant. Samples were immediately centrifuged and the plasma collected and stored at - 2 0 ' C until utilized. Slides coated with 3ml of l~°J, ~, agar in Veranol buffer were used for all immunological experiments. Antigenic cross reactivity was determined using the double immunodiffusion technique of Ouchterlony (I 948). Immunoelectrophoretic assays (Scheidegger, 1955) were run at 8 mA/slide, 150V, for 70min, using 0.075M cold Veranol buffer, pH 8.6.

MATERIALS AND METHODS The purification methods for Hp's from goats, sheep, cows and pigs used in the present study have been previously described by Travis & Sanders (1972). To obtain subunits for structural studies, lyophilized Hp was reduced and alkylated according to the method of Gordon et al. (1968), with slight modifications. 75 mg of lyophilized Hp were dissolved in 3 ml of 5M guanidine hydrochloride (GuHCI)/0.05M hydroxymethyl aminomethane (Tris), pH 7.8, and dialyzed overnight in the same buffer. To the 3 ml solution, 0.15 ml of 2-mercaptoethanol were added. This solution was then allowed to stand at room temperature for 3 hr before chilling to 4°C. Alkylation was achieved by the addition of 350 mg iodoacetamide in 1.5 ml (cold) 5M GuHCI/0.25M Tris, pH 8.2. This mixture remained at room temperature for 30 min before again dialyzing overnight in 500ml of 5M GuHCI/0.05M Tris, pH 7.8. To separate subunits, this solution was eluted through a 3 × 120cm column packed with Sephadex G-200 which had previously been equilibrated with 5M GuHCI/0.05M Tris, pH 7.8. The eluant was collected in approximately 2.5 ml fractions and analyzed to detect protein on a Beckman 25 UV spectrophotometer at 280nm. Fractions were then pooled into three volumes containing unreduced protein and separated subunits. These volumes were dialyzed against running water for 3 days, then lyophilized. Sodium dodecyl sulfate (SDS) gel electrophoresis was used for molecular weight determinations according to methods previously described (Weber & Osborn, 1969; McDonagh et al., 1972), along with gel filtration estimations (Andrews, 19641. The sodium phosphate buffer for the SDS gels (pH 7.2) contained 0.1~o SDS. Gels were 5.5~. Protein samples were prepared as follows: 10.0rag protein were added to 500 Z of a solution composed of 10ml (pH 7.2) sodium phosphate buffer solution, 0.1g SDS, and 0.2 ml 2-mercaptoethanol. The protein solutions were then incubated at 37°C for 3 hr. In some instances samples were incubated for 4 hr, with iodoacetamide added in the ratio of 12mg/10mg of protein for the last 2hr. Samples were then stored at -20°C. Prior to use, samples were reincubated for I hr at 37°C. Electrophoresis was car-

PEAKS:

RESULTS Column chromatography

Reduced and alkylated samples of goat, sheep and pig Hp's were subjected to G-200 gel filtration chromatography in order to demonstrate and obtain purified subunits. Cattle Hp was not available in sufficient quantity for this assay. Sheep proteins were used to calibrate the column and construct a molecular weight curve. Figure ! shows the profile obtained from sheep Hp. Unreduced protein is eluted as peak i, followed by separated subunits in peaks 2 and 3, Subunit molecular weights are indicated above their respective peaks. Goat and pig Hp's yielded similar elution profiles with peak 3, however, in both cases having a slightly different elution volume. Molecular weight estimations of goat Hp subunits yielded values of 43,000 and 20,000, while those of pig Hp were 43,000 and 13,000.

I

2

3 Sheep

1.50 1,25

==J

1.00 i

I EFFLUENT

1

J VOLUME

I

1 ''--.-J

ml

Fig. I. Sephadex G-200 chromatographic profile of sheep Hp.

Structure and evolution of Artiodactyla haptoglobins

391

Table 1. Molecular weights of Artiodactyla Hp subunits determined from SDS gel electrophoresis of the native protein* Treatment before electrophoresis Subunit

Reduced and alkylated

Reduced only (n (n (n (n

= = = =

Range

Goat 7 Sheep ~ Cow :t Pig ~

18.200 18,150 17.350 14,100

(n (n (n (n

= = = =

12) 7) 3) 5)

18,600 16,850 17.650 14,250

4) 4) 2) 3)

16,300-20,100 15,900-19,700 16,700-18,600 11,500--16,700

Goat fl Sheep fl Cow fl Pig [3

31.600 35.600 29,000 52,900

(n = (n = (n = 01 =

13) 2) 2) 5)

31,575 (n = 4) 35.250 (n = 2) -44,850 (n = 3)

28.300-35,200 32,600-38.600 44,300-55,300

* All numbers are the mean values of the number of assays indicated in parentheses.

Table 2. Molecular weights of Artiodactyla Hp subunits determined from SDS gel electrophoresis of subunits previously purified by column chromatography* Treatment before electrophoresis Subunit

Reduced and alkylated

Reduced only

Range

Goat ~ Sheep :t

16,500 (n = 1) --

17,200 (n = 2) 15,800 (n = 2)

16,500-17,500 15,740-15,800

Goat fl Sheep fl

28,850 (n = 1) --

-38,200 (n = 2)

-37,150-38.700

* All numbers are the mean values of the number of assays indicated in parentheses.

SDS gel electrophoresis Molecular weight determinations of the subunits from Artiodactyla Hp's were obtained from SDS gel electrophoretic experiments. Standard curves were prepared by measuring migration distances of known proteins. These included: myoglobin (17,500), sheep IgG light chain (25,000), pepsin (32,700), ovalbumin (45,000), sheep IgG heavy chain (50,000), bovine serum albumin (69,000), sheep transferrin (75,000), and in some instances, a dimer of ovalbumin (90,000). Molecular weight data are presented in Tables 1 and 2. Data from Table 1 were derived from whole protein that had either been reduced and alkylated or simply reduced. Subunit molecular weight determinations are comparable in both cases. Goat, sheep and cow or-like subunits all have molecular weights in the range of 16,000 to 18,500. Goat and cow t-like subunits have molecular weights of 31,500 and ~29,000, respectively, while that of sheep is slightly heavier at 35,000. Pig subunit data were most variable. Estimations from gel filtration column chromatography and SDS gel electrophoresis indicated a range for the ~-like subunit of 11,000 to 17,000, while the fl-like subunit falls in the range of 44,000 to 55,000. Data from Table 2 are derived from purified subunits obtained from column chromatography. Molecular weight determinations of goat and sheep subunits are in close agreement with those in Table 1.

Carbohydrate determinations The C H O content of the Hp's investigated is presented in Table 3. They are listed as per cent of total

glycoprotein by weight. Table 3 gives per cent C H O derived from purified proteins. Goat and cow Hp's have a C H O content of 3 ~ , while that of sheep is higher at 16~o. Data for the goat, cow and sheep were obtained from two assays, reflecting good precision in the results that are reported.

lmmunoelectrophoresis and immunodiffusion Figure 2 presents a comparison of Artiodactyla and human Hp mobilities following immunoelectrophoresis of partially purified fractions. Hp arcs were identified by staining the complete pattern for peroxidase activity. Mobilities of goat and sheep Hp's are identical, while cow, pig, and human Hp's are similar, demonstrating slightly greater mobilities than goat and sheep. All mobilities, however, are in the ct2-glo bulin range. Immunodiffusion experiments were performed in order to determine what degree of similarity exists for the antigenic determinant sites among the Artiodactyla and human Hp's. F r o m slides A and B (Fig. 3) it can be seen that anti-goat, anti-sheep and anticow Hp's each exhibit complete cross reactivity with goat, sheep and cow Hp's, but do not demonstrate Table 3. Carbohydrate content of intact Artiodactyla Hp's Hp source Goat Sheep Cow

CHO/Glycoprotein (mg/mg)

Per cent

0.0325 0.1604 0.0312

3 16 3

F

E

D

C

13

A

Fig. 2. Immunoelectrophoretic patterns illustrating human and Artiodactyla Hp mobilities. (A) human plasma cross reacted against human anti-serum; (B), (C), (D), and (E) are goat, sheep, cow and pig Hp fractions respectively, which were cross reacted against their own anti-sera; (F) human plasma cross reacted against human anti-Hp serum. Arrows indicate Hp arcs.

o

©

.j

e'~

.<

-r

7~

C

3

C

O

Os

G goat ¢x o~subunit /3/3 subunit H human pldsma P pig S sheep

cow

pO

HO

s/30

~O

G~O

cO

Oc PO

O G

Fig. 3. Immunodiffusion of various anti-Hp sera with indicated Artiodactyla Hps and human plasma. Artiodactyla samples were solutions of 5 mg protein/'l ml dHOH.

Ip

~lp

HO

%

D

G

P

D

S

OG ~so~

~Go~

Os

O OG

S P

G

O G

cO O OP

:O

C

HO

OG so ~ O

O S

cO O OP

sO~

HO

S

O

G

O

w.

O

,--1

>

O

E"

o

e-,

¢..

394

WALKER H. BUSBY JR and JAMES C. TRAVIS Table 4. Immunological cross reactivity of Artiodactyla and h u m a n Hp's

Antigens Antisera Anti-goat Hp Anti-sheep Hp Anti-cow Hp Anti-pig Hp Anti-human Hp

Goat Hp

Sheep Hp

Cow Hp

Pig Hp

C C C X X

C C C X X

C C C X X

X X X C X

Human plasma P P P X (.......

Cross reactivity: C = complete, P = partial, X = none, and

cross reactivity with pig Hp. Anti-pig Hp reacts only with pig Hp. However, anti-goat Hp exhibits cross reactivity with human serum. Anti-sheep and anticow Hp's in subsequent experiments have also demonstrated some slight cross reactivity with human serum. Furthermore, cross reactivity between antigoat Hp and either goat Hp subunit (slide C) is not observed. Results from these and other slides are compiled in Table 4.

Goat ~

Goat [~

X

X

. . . .

= not assayed.

whose bands appeared particularly sharp and distinct. Sheep /~ subunit was found to have a molecular weight of 38,0(~-somewhat higher than goat and cattle. This value came from two gels, the samples being gel filtration purified subunits, with reduction only prior to application. ,q subunit estimations from whole sheep Hp were less accurate, being derived from bands appearing lighter and more diffuse. The molecular weight of pig/~ subunit reported in Table 1 should be viewed with caution. Pig Hp, being monomeric, may be contaminated with IgG (molecuDISCUSSION lar weight 150,000), which elutes with Hp during gel The demonstration of naturally occurring poly- filtration purification. Further complications arise as meric Hp's in certain Artiodactyla has led to their IgG exhibits subunit structure similar to that of Hp, comparison with man, who until recently was thought with weights of 50,000 and 25,000 for the heavy and to be unique among mammals in exhibiting a poly- light subunits, respectively. Pig /~ subunit molecular meric Hp. It has been postulated that the alleles re- weight determinations were consequently difficult, sponsible for encoding Hp in these species have arisen with the results inconclusive from both gel filtration from common :~l-like and ,6 ancestral genes, and that and SDS, Of major importance, however, was the :~ the genetic mechanism resulting in the formation of subunit molecular weight determination for pig Hp the ~2 allele in humans has been closely paralleled which was approximately 13,000. This value, indicatin Artiodactyla evolution (Travis & Sanders, 1972). ing an ~l-likeness, is not clouded by IgG contamiFurther structural evidence is presented here to sup- nation. port this hypothesis. CHO content for goat and cow Hp's were both Molecular weight estimations from G-200 gel filtra- low (3~o), while sheep exhibited a higher value of 16%. tion column chromatography of reduced and alkyl- Moretti & Donati (1973) have reported a sheep CHO ated sheep, goat and pig Hp's yield similar /~ subunit content of 26.9~o (4.49/00 of this value, however, repvalues (42,01~-43,000) but dissimilar :~ subunit values resents amino sugars, which are not detected by the (13,000-20,000). In humans, subunit molecular assay used in the present study). weights for/3, ~1, and ~2 are 40,000, 9000 and 17,300, The seemingly incongruent datum concerning the respectively (Barnett et al., 1972; Smithies et al., 1962; CHO content of sheep Hp (Table 3) can be accounted Connell et al., 1966). Polymeric capabilities are con- for in two ways. Firstly, if one compares molecular ferred upon human Hp by the Hp ~2 allele, encoding weights of cow, goat and sheep proteins, one sees a the :~2 polypeptide chain (Bearn & Franklin, 1958; uniform ~ subunit weight, while the /~ subunit of Allison, 1959). One would assume, therefore, that goat sheep is larger than that of goat and cow by and sheep exhibiting polymeric Hp would have c~ 6000-9000. This difference could be attributed to subunits weighing approximately 17,500 (c~2-1ike), CHO content. Secondly, goat/~ and ~ subunit weights while pig Hp, being monomeric, would have an of 39,000 and 18,500, respectively, have been reported subunit with a molecular weight closer to 9000 (Travis et al., 1975). This, together with a reported (:~-like). The validity of these assumptions is sug- CHO content of 19-23~o (Travis et al., 1975) for gested by the fact that goat and sheep were found Table 5. Molecular weights of Artiodactyla subunits to have ~ subunits in the range 16,000 to 19,000, while that of pig was 13,500. 17,000 SDS gel electrophoresis molecular weight deter- Goat ~ Sheep :~ 16,500 minations were compatible with gel filtration estima- Cow ~t 17,500 tions but with lower values. The data compiled in Pig ~ 13,500 Tables 1 and 2 represent molecular weights of Goat /3 31,600 subunits, the formation and treatment of which, prior 38,000 to electrophoretic application, were experimentally Sheep /3 Cow /3 29,000 different. The most reliable molecular weights of Pig [3 subunits (Table 5) were determined from selected gels

Structure and evolution of Artiodactyla haptoglobins

395

NON-POLYMERIC HP

POLYMERIC HP

POLYMERIC HP

I

RAT

~

I

ICR.t

ICRm A-RABBIT "MONKEY HUMAN WHALE

A

CAT

DOG

HORSE

PIG

GOAT BISON ELK SHEEP CATTLE DEER

I

HP~ BOVIDAE

I

RODENTIA LAGOMORPHA PRIMATES CETACEA

CARNI VORA

1 ANCESTRAL

PERI SSODACTYLA

CERVIDAE

[HP~

I

I

ART IODACTYLA

I

MAMMALS

Fig. 4. Diagram illustrating the evolutionary relationships of certain mammals with the information from Hp studies indicated. ICR*= immunologic cross reactivity with anti-human Hp sera, while ICR** = immunologic cross reactivity with anti-goat Hp sera (Travis & Sanders, 1972).

whole glycoprotein, gives an adjusted polypeptide weight for the goat fl subunit of 31,200-32,800 (Travis et al., 1975). Sheep Hp used in the present experiments came from a parallel purification with the goat Hp used by those researchers. Assuming that much of the CHO moiety of the sheep Hp is located on the fl polypeptide chain (the entire CHO moiety of human Hp is located on the fl subunit--Shim & Bearn, 1964; Cheftel & Moretti, 1966), an adjusted polypeptide weight for the sheep fl chain would approximate 30,000-33,000. This estimation would then be compatible with the goat and cow subunit molecular weights, as indicated in Table 5. It should be noted that the goat and cow Hp used in the present study came from subsequent purifications. This suggests a possible loss of CHO from non-identical purification procedures. Reconciling CHO data with SDS data gives similar molecular weight values for the polypeptide moieties of the fl and c( subunits of goat, sheep and cow, which would be approximately 31,000 and 17,000, respectively. Immunological studies allow protein comparisons based on electrophoretic mobilities and antigenic cross reactivities. The mobilities of the various Artiodactyla Hp's, as ascertained from immunoelectrophoretic experiments, are all in the ~2-range. This indicates a similarity in net charge and presumably in amino acid content of the proteins. Immunodiffusion experiments, in which specific antisera are cross reacted with various Hp's, provide a means for comparison of the structural antigenic sites on these proteins. Experiments in this study indicate structural homology between the polymeric Hp systems of goat, sheep and cow, as they are completely cross reactive among themselves, while the monomeric Hp system of pig exhibits no homology with the other Artiodactyla. Furthermore, the antigenic sites do not appear to be located solely on either subunit, since no cross reactivity was observed between either goat subunit and anti-goat Hp. This would indicate that a specific stereochemical interac(',ILl'. 60 ,'41~

I)

tion between subunits is necessary for the manifestation of antigenicity. From these immunodiffusion experiments, the demonstration of partial cross reactivity between antigoat Hp and human serum is reported. Further data to clarify this observation have been presented in another report (Travis, 1977). A schematic of mammalian evolution according to Travis & Sanders (1972) is presented in Fig. 4. Probable points of genetic duplication resulting in the Hp ~t allele are indicated. Data gathered in this study support the postulate that an ctZ-like allele exists in certain Artiodactyla as the result of a similar but independent genetic event as that occurring in human Hp evolution. Structurally, the c( peptides of goat, sheep and cows resemble the ~(2 peptide of humans, while that of pig exhibits properties intermediate between human ct1 and ct2 peptides. As pig Hp is monomeric, and goat, sheep and cow Hp's are polymeric, the view that the ~(2 peptide initiates polymerization is supported. Acknowledgements--This work was supported by grants from the UNCC Research Foundation and the United Medical Research Foundation of North Carolina.

REFERENCES

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396

WALKER H. BUSBY JR and JAMES C. TRAVIS

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