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The structure of bovine secretory component
Veterinary Immunology and Immunopathology, 17 (1987) 37-49 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
37
THE STRUCTUREOF BOVINE SECRETORYCOMPONENT
D. BEALE Agricultural and Food Research Council, I n s t i t u t e of Animal Physiology and Genetic Research, Babraham, Cambridge, CB2 4AT, U.K.
ABSTRACT Beale, D., 1987. The structure of bovine secretory component. Vet. Immunol. Immunopathol., 17: 37-49. Bovine secretory component (SC) has been cleaved with trypsin into a series of fragments and their N-terminal amino acid sequences have been determined. The close homology with the known sequence of human SC has enabled the sequential order of the fragments to be deduced. The results indicate that bovine SC cQnsists of a single glycosylated polypeptide chain (Mr 74,000) folded into five globular immunoglobulin-like domains. A protein (Mr 94,000) has been isolated from detergent solubilised bovine epithelial membranes from l i v e r , intestine and mammary gland. This membrane protein is specific for the binding of J-chain linked IgM and IgA dimers. I t can be proteolytically cleaved into a water soluble SC-like portion and a detergent soluble hydrophobic portion. Bovine SC is therefore most l i k e l y to be the extracellular part of an e p i t h e l i a l receptor which mediates the transport of IgA dimers to mucosal surfaces. The various t r y p t i c fragments from bovine SC have been shown to d i f f e r in t h e i r relative binding a f f i n i t i e s for IgM and IgA dimers. The results imply that the f i r s t three domains of bovine SC are most involved in binding and domains 4 and 5 play subsidiary roles. Computerized prediction and modelling methods have been used to deduce possible t e r t i a r y and quaternary structures for SC. There are good indications t h a t the molecule has an elonaged "zig-zag" s t r u c t u r e s t a b i l i z e d by longitudinal inter-domain contacts. A model of SC bound to IgA dimer is presented. INTRODUCTION During a study of immune globulins in bovine milk Groves and Gordon (1967) isolated a previously undetected protein which they called glycoprotein-a.
It
was subsequently identified by Butler (1971) as bovine secretory component (SC) which had been reported by Mach et al. (1969) and Porter and Noakes (1970) to be present in bovine external secretions both in the free Form and as a component of SIgA. Physicochemical studies on bovine SC (Butler, 1971; Labib et a l . ,
1976) established i t
as a rather elongated molecule consisting of a
single glycosylated polypeptide chain of Mr 70-79,000.
One mole of SC was
found per mole of SIgA. A series of reports from a number of laboratories working with human and rabbit SC gradually established that the protein formed, or was related to, an 0165-2427/87/$03.50
© 1987 Elsevier Science Publishers B.V.
38 epithelial
receptor which mediates the t r a n s c e l l u l a r transport
of J-chain-
linked IgM and IgA polymers to mucosal surfaces (Brandtzaeg, 1985; Underdown and S c h i f f , 1986). My laboratory began to study bovine SC in 1984 with the i n i t i a l
aims of ( i )
locating the binding s i t e , (2) establishing the presence of a bovine epithelial polyimmunoglobulin receptor and (3) determining the primary structure and deducing secondary, t e r t i a r y and quaternary structures.
Progress towards the
f i r s t objective was made by preparing a t r y p t i c Fragment, of Mr 25,000, which binds polymeric immunoglobulin (Beale and Hopley, 1985) and locating i t s position in the SC molecule as described here.
The second aim was achieved by
isolating a protein from detergent solubilized epithelial membranes which bound polymeric immunoglobulin and had a structure similar to that of bovine SC (Beale and Hopley, 1986). The third aim was greatly helped when Mostov et al. (1984) published the amino acid sequence for the rabbit polyimmunoglobulin receptor, translated from a cDNA sequence, and E i f f e r t et al. (1984) published the sequence of human SC obtained directly from the protein.
These sequences
indicated that SC and the extracellular part of the receptor have five homology regions related to
immunoglobulin
domains.
We carried out computerized
secondary structure predictions on these sequences, deduced possibly t e r t i a r y structures and tested the v a l i d i t y of these by locating accessible and inaccessible sites for t r y p t i c cleavaje in bovine SC (Beale and Coadwell, 1986; and this paper). MATERIALS AND METHODS Preparation of bovine SC Milk
whey p r o t e i n s
were
fractionated
to
obtain
SC by ion-exchange
chromatography and gel F i l t r a t i o n as described by Butler (1971) and Labib et al. (1976). Preparation of bovine epithelial pol~immunoglobulin receptor The method of Kuhn and Kraehenbuhl (1979, 1981) as modified by Beale and Hopley (1986) was followed. Epithelial membranes from bovine l i v e r , intestine and mammary gland was solubilized in detergent and subjected to a f f i n i t y chromatography with rabbit IgG-Sepharose, rabbit
IgG - (anti-bovine SC)-
Sepharose and human IgM-Sepharose. Fragmentation with trypsin Solutions of protein (0.5%) in 0.15M NaCl, O.1M Tris-HCI, pH 8.0, were digested with trypsin (protein/enzyme ratio 100:1) For various times at 37oc. Then trypsin inhibitor at twice the weight of trypsin was added and the samples stored at -20oc.
39
High performance l i q u i d chromatography An LKB TSK G3OOOSWUltropac gel f i l t r a t i o n column (7.5 x 600 mm) was used in l i n e with an LKB 2150 HPLC pump, a Rheodyne 7125 injector, a Pye Unicam PU4020 UV detector and a Philips PM 8252 recorder.
Elution was carried out with 60mM
phosphate buffer, pH 7.4, in most cases, and the eluate monitored by absorption at 280 nm. SDS pol~acr~lamide gel electrophoresis The method of Laemmli (1970) as modified by Richardson and Feinstein (1978) was employed.
10% and 12% w/v polyacrylamide gels with 3% w/v stacking gels
were used with a discontinuous T r i s - g l y c i n e buffer containing 0.1% SDS. Samples were boiled in 1% SDS with or without mercaptoethanol.
Protein was
detected with Coomassie B r i l l i a n t Blue or by the s i l v e r stain method of Oshawa and Ebata (1983).
Stained gels were scanned d e n s i t o m e t r i c a l l y .
Protein
labelled with 1251 was detected by autoradiogrphy or by s l i c i n g and counting gels. Relative binding a f f i n i t ~ This was measured using the i n h i b i t i o n method of Socken and Underdown (1978) as modified by Beale and Hopley (1985).
Serial dilutions of the test protein
were mixed with a standard amount of 125 I-SC and the amount of radioactivity bound to a standard amount of IgM-Sepharose under standard conditions was measured. N-terminal amino acid sequencing This was carried out on an Applied Biosystems 470A Gas Phase Protein Sequencer in series with an Applied Biosystems 120A PTH Analyzer. RESULTS Fragmentation of free bovine SC Figure la shows the results of running reduced aliquots of t r y p t i c digests of bovine SC on SDS polyacrylamide gel electrophoresis. as densitometric scanning profiles.
They are represented
I t w i l l be seen that as digestion time
lengthens the main products occupy lower positions on the gels thereby i n d i c a t i n g lower molecular weights. themselves in groups on the gels.
Also the products tend to position
Fragmen%s Q and R, present at an early stage
of the digestion, run near the position of an Fdu' marker which consists of three immunoglobulin domains, two of them glycosylated.
Fragments S and T,
which appear at an early stage, and fragment A, which appears at a later stage, a l l run near the position of an Fdu marker which consists of two immunoglobulin domains, one of them glycosylated.
Fragments B and C, which appear at a late
40
-Fd~
R a
s
-Fdju
>
1
-p2m o
2
5
10
15
25
40
60 min
b
Figure 1.
Densitometric scanning p r o f i l e s from SDS polyacrylamide gel electrophoresis of a) reduced and b) unreduced aliquots of 0-60 min t r y p t i c digests of bovine SC. The positions of Fd'u, Fdu and B2M markers are indicated on the right.
stage of the digestion, run near to the position of B2-micro-globulin which consists of one immunoglobulin-like domain. Figure lb shows the results of running similar aliquots unreduced on SDS polyacrylamide gel electrophoresis.
It
is obvious that fragments Q, S and T
are now absent but there is more material in the position of SC.
This suggests
that fragments Q, S and T are only freely released by reduction. The interpretation of the fragmentation results is that bovine SC consists of f i v e globular immunoglobulin-like domains in accordance with the published sequence information
For human SC ( E i f f e r t
et
al.,
1984) and r a b b i t
polyimmunoglobulin receptor (Mostov et a l . , 1984). I n i t i a l t r y p t i c cleavage of SC produces a fragment Q, of three domain size, disulphide bridge to fragment S or T, of two domain size. degree of glycosylation.
Fragments S and T probably only d i f f e r in t h e i r Cleavage near to the disulphide bridge then freely
releases fragment R, of three domain size but s l i g h t l y smaller than fragment Q. Fragment R eventually gives rise to the two-domain fragment A.
Fragments B and
C are derived by further cleavage of both fragments S and T. Figure 2a shows the 280 nm absorption profiles from the HPLC of t r y p t i c digests of bovine SC.
Figure 2b shows the results of running reduced aliquots
41
elution .-.z,
SC
20min
A
C
peak SC
0
R
A
c
B
-68,ooo -43,000
-23,000
Figure 2.
a) 280 nm absorption profiles from gel f i l t r a t i o n HPLC of tryptic digests of bovine SC. b) Densitometric scanning profiles from SDS polyacrylamide gel electrophoresis of reduced aliquots of HPLC peaks shown in a).
of the HPLC peaks on SDS polyacrylamide
gel
electrophoresis.
represented as densitometric scanning p r o f i l e s .
They are
I t was found that peak R from
the I0 min digest consisted almost e n t i r e l y of fragment R of Mr 42,000.
Peaks
A, B and C from the 35 min digest contained single components (fragments A, B and C) of Mr 25,000, 14,000 and 12,000 respectively. N-terminal amino acid sequences of t r ~ p t i c fraQments These results are shown in Figure 3 where they homologous
sequences from human SC and rabbit
are
aligned
Ig receptor.
with
the
Computerized
sequence searching showed that these are the only s i g n i f i c a n t alignments. It
will
be seen t h a t
the
N-terminal
sequence of
i d e n t i c a l with the N-terminal sequence of the f i r s t
fragment R is
almost
homology region of human SC
42 bovine SC, fragment Q, bovine SC. fragraent R, human SC, ]st homology region, rabbit receptor, ]st honmlogy region,
N-terminus. N-terminus. N-terminus. N-terminus.
SPIFGPEEVD SPIFGPEEVDSVEGSSVSITC KSPIFGPEEVNSVEGNSVSITC PSSIFGPGEVNVLEGDSVSITC
bovine SC, fragment A, N-terminus. hu~an SC, Ist homology region, C-terminus. rabbit receptor, Ist homlogy region, C-terminus.
ELNFQVSLEVSQ RGLSFDVSLEVSQ RGLDFGVNVLVSQ
bovine SC, fragment B, N-terminus. human SC, 4th homology region, mlddTepert. rabbit receptor, 4th homOlogy region, mlddlepart.
LALLTVPGPVNYKVILNQLTDQDTGFYWC RLSLLEEPGNGTFTVILNQLTSRDAGFYWC RLALFEEPGNGTFSVVLNQLTAEDEGFYWC
bovine SO, fragment C, N-termlnus. ,,uman SO, 5th homo]ogy region, N-termlnus. rabbit receptor, 5th homology region, N-terminus.
VPLGEFSLKVPKVVDAWLSEPLKLSCYFPPKKYWFF KIIEGEPNLKVPGNVTAVLG£TLKVPCHFPCKFSSY£ QIVDGEPSPTIDK-FTAVQGEPVEITCHFPCKYFSSE
Figure 3.
and d i f f e r s
N-terminal amino acid sequences of t r y p t i c fragments from bovine SC compared with homologous sections from human SC ( E i f f e r t et a l . , 1984) and rabbit receptor (Mostov e t a l . , 1984).
at only one position from the N-terminal
sequence of bovine SC
published by Labib et a l . (1976). The N-terminal sequence of fragment A is very homologous with the C-terminal sequences of the f i r s t
homology regions of human SC and rabbit receptor.
The
homologous sections are immediately preceded by arginine residues which would provide poteqtial sites for t r y p t i c cleavage. The N-terminal
sequence of
fragment 3 is
very homologous with the middle
parts of the fourth homology regions of human SC and rabbit receptor. each case the
homologous
section
is
immediately preceded
Again in
by an arginine
residue. The N-terminal amino acid sequence of fragment C is very homologous with the N-terminus of the f i f t h is
homology region of human SC where the relevant section
immediately preceded by a l y s i n e residue which would provide a potential
s i t e for t r y p t i c cleavage. The fragmentation process The results
of t r y p t i c
fragmentation and N-terminal sequence analysis are
summarized d i a g r a m m a t i c a l l y
in
Figure
4.
The f i v e
domains
of
SC are
represented as loops, each containing an intra-domain disulphide bridge as in immunoglobulin domains.
Initial
tryptic
cleavage occurs w i t h i n domain 4 and
gives rise to fragment Q disulphide bridge to fragment T by the intra-domain bridge of domain 4.
Fragment Q consists of domains I ,
terminal portion of domain 7. domain 4 plus most i f
not a l l
2 and 3 plus an N-
Fragment T consists of a C-terminal portion of of domain 5.
Further cleavage near the N-
terminus of domain 4 then releases fragment R which consists of domains 1, 2
43 R
NH2
I COOH
Q
Figure 4.
and 3.
T
-
-
Diagrammatic representation of the t r y p t i c fragmentation of bovine SC. The SC polypeptide chain is shown as consisting of f i v e domains, represented as loops, each containing an intra-domain disulphide bridge.
Cleavage towards the C-terminus of domain 1 then gives rise to fragment
A which consists of domains 2 and 3 plus a portion of the C-terminal end of domain 1.
Further cleavage within domain 4 and at i t s C-terminus gives rise to
fragment B, which consists of a C-terminal section of dQmain 4 plus most i f not all of domain 5, and C, which consists of domain 5. Relative binding a f f i n i t i e s of t r y p t i c fragments The inhibition results are shown graphically in Figure 5. fragment
I t is clear that R (domains 1, 2 and 3) inhibits the binding of 1251-SC to IgM-
7~
.
.
.
3O
,
2
|
4
,
, ,
6
, ,
8 10
30
60
100
po
Figure 5.
Inhibition of 1251-SC binding to IgM-Sepharose. • R, ~ fragment A, A Fragment B, ~ Fragment C.
SC, V fragment
44 Sepharose to a similar extent as bovine SC does. Fragment A (domains 2 and 3) also i n h i b i t s
but
is
considerably less effective than SC or fragment R.
Fragment B (part of domain 4 plus domain 5) and fragment C (domain 5) do not inhibit. These results imply that SC domains I and 2 are most involved in binding polymeric immunoglobulin, domain 3 is also l i k e l y to be involved but domains 4 and 5 probably play subsidiary roles. Fragmentation of bound SC Figure 6 shows the results of digesting SIgA (bovine SC bound in vivo to IgA dimers) and 1251-SC bound
in v i t r o to
IgM and IgA dimers.
scanning profiles and radioactivity profiles are shown.
It
Densitometric will
be seen
(Figure 6a,b) that even after 2 hr digestion of SIgA, fragment ~ is s t i l l present in good yield whereas only a 1 hr digestion of unbound SC degrades i t to Fragments A, B and C (Figure 6c,d).
Similar results were obtained when
1251-SC was bound in v i t r o to IgM or IgA dimers (Figure 6e-h) except that some Fragment R was present as well as Fragment Q. be released from digested SIgA by reduction.
Further, Fragment Q could only I t has the same N-terminal amino
acid sequence as Fragment R (Figure 3). These results imply that during the binding of polymeric immunoglobulin in vivo the potential t r y p t i c cleavage sites near the C-terminus of domain 1 an the N-terminus oF domain I become burised and a disulphide bridge Forms between IgA and domain I , 2 or 3. v i t r o but there is l i t t l e
a b SC
Figure 6.
A very similar burying of cleavage sites occurs in i f any disulphide bridging.
C
H
sI
f
g
h
SCl I
Fragmentation of bovine SC bound in vivo and in v i t r o . Unfilled profiles represent densitometric scans of SDS polyacrylamide gel electrophoresis of reduced samples. Filled profiles represent radioactivity of gel slices and are shown on the r i g h t of the respective densitometric scan. SIgA, a) undiqested and b) digested 2h. SC, c) undigested and d) digested lh. 1251-SC bound in v i t r o to IgM, e) undigested and f) digested 2 h. 1251-SC bound in v i t r o to IgA dimers, g) undigested and h) digested 2h.
45
~'
Figure 7.
NH 2
Diagra~natic representation of the immunoglobu!in-like SC domain. B-strands are shown as long arrows. Connecting sequences between strands are shown as l i n e s . The four shaded B-strands make ;~p one B-shee and the f i v e ,Jnshaded B-strands :hake up the other B-sheet. SC domains 1-4 have t e r t i a r y structures similar to that shown but domain 5 has one B-strand less in each sheet. Short arrows indicate potential sites for t r y p t i c hydrolysis which are cleaved in one or other of the bDvine SC domains. Black diamonds indicated potential sites for t r y p t i c hydrolysis which are not cleaved in '~n~'~qe SC.
Accessible and inaccessible sites for tr~ptic hydrol~sis in the t e r t i a r ~ structural models of SC domains Figure 7 is a diagra,mnatical representation of the t e r t i a r y structures of SC domains 1-4 computed in my laboratory (Beale and Coadwell, 1986).
They have
many of the characteristics of immunoglobulin domains and consist of a f i v e stranded B-sheet and a four-stranded B-sheet joined by an intra-domain disulphide bridge. The t e r t i a r y structure of domain 5 is very similar but has two less B-strands. It
will
be seen that
potential
actually cleaved in bovine SC al] positions.
sites lie
for t r y p t i c hydrolysis that are
outside the B-strands at exposed
In contrast, the potential sites For t r y p t i c hydrolysis that are
not cleaved in bovine SC a l l positions.
lie
w i t h i n the B-strands
at inaccessible
Fragmentation of bovine epithelial pol~immunoglobulin receptor In these experiments a mixture of 1251-receptor and unlabelled SC was digested with trypsin in 0.1% detergent and reduced aliquots were run on SDS polyacrylamide gel electrophoresis. The results are shown as densitometric scans and radioactivity profiles in Figure 8.
46 a
p
04,00074,000-
P
m
Figure 8.
Fragmentation of a mixture of bovine SC and 1251 labelled bovine epithelial polyimmunoglobulin receptor. Unfilled profiles represent densitometric scanning p r o f i l e s from SDS polyacrylamide gel electrophoresis of reduced samples. F i l l e d p r o f i l e s represent r a d i o a c t i v i t y of gel slices and are shown on the right oF the respective densitometric scans, a) undigested mixture, b) 10 min digest, c) 25 min digest, d) 40 min digest. Molecular weights are shown on l e f t ,
a
b
iD
Figure 9.
Phase separation with Triton X-114 of bovine e p i t h e l i a l receptor and t r y p t i c fragments. The profiles represent r a d i o a c t i v i t y of SDS polyacrylamide gel slices of reduced samples, a) undigested sample, detergent phase on l e f t and aqueous phase on right, b) sample digested 20 min, detergent phase on l e f t and aqueous phase on right.
In the undigested mixture (Figure 8a)most of the r a d i o a c t i v i t y was detected at a position indicative of Mr 94,000 although appeared at Mr 74,000 coincident with SC.
a significant
amount also
As digestion proceeded (Figure 8b-d)
most of the r a d i o a c t i v i t y occupied positions of decreasing molecular weight that were exactly coincident with those of SC fragments. When the experiments were repeated using phase separation in Triton X114 (Bordier, 1981) the r a d i o a c t i v i t y in the undigested mixture was found mainly in the detergent phase (Figure 9a) but after t r y p t i c digestion of the mixture nearly all the r a d i o a c t i v i t y appeared in the aqueous phase (Figure 9b).
47 DISCUSSION The experiments described in this paper provide good evidence that bovine SC is derived from an epithelial membrane receptor which mediates the transport of IgM and IgA polymers across the epithelial layer to the mucosal surface.
The
extracellular part of the detergent solubilized receptor is easily identical with SC.
The presence of SC in SIgA is presumably an aftermath of the
transport process. The membrane protein appears to be the bovine equivalent of the human polymeric immunoglobulin receptor reported by Mostov and Blobel (1982) and the rabbit polymeric immunoglobulin receptor described by Kuhn and Kraehenbuhl (1981) and Mostov et al. (1984). The bovine epithelial receptor and SC can be cleaved by trypsin into a series of well characterized fragments whose locations in the SC molecule have been determined by N-terminal sequence analysis.
Fragments obtained from human
SC by Kobayashi et al. (1973) but not f u l l y characterized are probably the human equivalents of the bovine SC fragments. The physicochemical properties of the t r y p t i c fragments support the concept of a five domain SC molecule put forward by Mostov et al. (1984) and E i f f e r t et al. (1984) based on internal sequence homologies. The fragments have different binding a f f i n i t i e s which suggest that the f i r s t three domains of SC are most involved in binding polymeric immunglobulin.
The r e l a t i v e proteolytic
resistance of the f i r s t three domains when SC is complexed with IgM and IgA polymers indicates that there are very close contacts between receptor and ligand (Beale, 1985). The locations of accessible and inaccessible sites for t r y p t i c hydrolysis in the bovine SC molecule support putative t e r t i a r y structures for the SC domains (Beale and Coadwell, 1986) which are very immunoglobulin-like as predicted by Mostov et al. (1984) and E i f f e r t et al. (1984). However, possibly quaternary structures derived by assembling the domains do not allow close inter-domain contact as found in immunoglobulin molecules. Experimental support is provided by the fact that we could not detect any significant interactions between the various Fragments of bovine SC. The main interdomain contacts which determine the quaternary structure are therefore l i k e l y to be weak longitudinal ones. T h i s leads to two quaternary structural models.
One has a horseshoe shape but would need an inter-domain
disulphide bridge between domains 1 and 5 to s t a b i l i z e the structure. I n i t i a l l y we thought that such a bridge might exist (Beale and Hopley, 1985) but have been unable to substantiate this experimentally. The alternative model which we now favour has a zig-zag shape which does not require a disulphide bridge and a similar structure for human SC has been proposed by Pumphrey (1986). A diagrammatical representation of the bovine SC molecule is
48
i
a
b
c
Figure 10. a) Putative quaternary s t r u c t u r e f o r bovine SC. The f i v e immunoglobulin-like domains are represented as c y l i n d e r s which interact l o n g i t u d i n a l l y to form a zig-zag structures, b) A similar representation of the Fc regions of two IgA or IgM monomers linked by J-chain which is shown as an oblong box. c) Diagrammatic representation of bound SC. Domain 1 is interacting with a CH2 domains of one of the Fc monomers and probably forms a disulphide bridge (not shown). Domain 3 is interacting with the CH3 domain of the other Fc monomer but would not Form a disulphide bridge. Domain 2 is interacting with J-chain. d) SC bound to an a l t e r n a t i v e arrangement of the Fc regions of two IgA monomers. The interactions are similar to those in c).
given in Figure 10 and the binding of J-chain-linked IgA dimers represented.
is
also
The domains are i l l u s t r a t e d as cylinders following the convention
of Schiffer et al. (1973) for immunoglobulin domains. Within each cylinder the peptide chain is folded into two B-pleated sheets linked by the intra-domain disulphide bridge.
REFERENCES Beale, D., 1985. Differences in fragmentation between bound and unbound bovine secretory component suggest a model for i t s interaction with polymeric immunoglobulin. Biochem, J., 229: 759-763. Beale, D. and Coadwell, J., 1986. Tertiary structures for the extracellular domains of the epithelial polyimmunoglobulin receptor (secretory component)
49
derived by primary structure comparisons with immunoglobulins. Comp. Biochem. Physiol. (in press). Beale, D. and Hopley, J.G., 1985. Fragmentation and reduction of bovine secretory component. Preparation of a biologically active fragment and some evidence for a multiple-domain structure. Biochem. J., 226: 661-667. Beale, D. and Hopley, J.G., 1986. A bovine epithelial membrane protein that binds polymeric inmunoglobulin and has a structure related to that of bovine secretory component. Biochem. J., 233: 37-40. Bordier, C., 1981. Phase separation of integral membrane proteins in Triton X114. J. Biol. Chem., 256: 1604-1607. Brandtzaeg, P., 1985. Role of J chain and secretory component in receptormediated glandular and hepatic transport of immunoglobulins in man. Scand. J. Immunol., 22: 111-146. Butler, J.E., 1971. Physicochemical and immunochemical studies of bovine IgA and glycoprotein-a. Biochim. Biophys. Acta., 251: 435-449. Eiffert, H., Quentin, E., Decker, J., H i l l m e i r , S., Hufschmidt, M., Klingmuller, 0., Weber, M.H.. and Hilschmann, N., 1984. The primary s t r u c t u r e of human free secretory component and the arrangement of disullphide bonds. Hoppe-Seyler's Z. Physiol. Chem., 365: 1489-1495. Groves, M.L. and Gordon, W.G., 1967. Isolation of a new glycoprotein-a and a globulin From individual cow milks. Biochemistry, 6: 2388-2394. Kobayashi, K., Yaerman, J.P. and Hereman, J.F., 1973. Studies on human secretory IgA ( I I ) . Comparative studies on a fragment of secretory component derived from secretory IgA and fragments obtained by enzymatic digestion of free secretory component. Immunochemistry, I0: 73-80. Kuhn, L. and Kraehenbuhl, J.P., 1979. Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells. J. Biol. Chem., 254: 11072-11081. Kuhn, L. and Kraehenbuhl, J.P., 19981. The membrane receptor for polymeric immunoglobulin is structurally related to secretory component. Isolation of membrane secretory component frown rabbit liver and mafmnary gland. J. Biol. Chem., 256: 12490-12495. Labib, R.S., Calvanico, N.J. and Tomasi, T.B., 1976. Bovine secretory component. Isolation, molecular size and shape, composition, and NH2terminal amino acid sequence. J. Biol. Chem,, 251: 1969-1974. Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Mach, J.P., Pahud, J.J. and Is]iker, H., 1969. IgA with secretory piece in bovine colostrum and saliva. Nature, 223: 952-954. Mostov, K.E. and Blobel, G.A., 1982. A transmembrane precursor of secretory component. The receptor for t r a n s c e l l u l a r transport of polymeric immunoglobulins. J. Biol. Chem., 157: 11816-11821. Mostov, K.E., Friedlander, M. and Blobel, G., 1984. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulinlike domains. Nature, 308: 37-43. Ohsawa, K. and Ebata, N., 1983. Silver stain for detecting lO-femtogram quantities of protein after polyacrylamide gel electrophoresis. Analyt. Biochem., 135: 409-415. Porter, P. and Noakes, D.E., 1970. Immunoglobulin IgA in bovine serum and external secretions. Biochim. Biophys. Acta, 214: 107-116. Pumphrey, R.S.H., 1986. Computer models of the human itnmunoglobulins: binding sites and molecular interactions. Immunology Today, 7: 206-211. Richardson, N.E. and Feinstein, A., 1978. Mouse i n t r a c e l ] u l a r IgM. Structure and identification of a free thiol group. Biochem. J., 175: 959-967. Socken, D.J. and Underdown, B.J., 1978. Comparison of human, bovine and rabbit secretory component-immunoglobulin interactions. Immunochemistry, 15: 499506. Schiffer, M., Girling, R.L., Ely, K.R. and Edmundson, A.B., 1973. Structure of a -type Bence Jones protein at 3.5A resolution. Biochemistry, 23: 46204631. Underdown, B.J. and Schiff, J.M., 1986. Immunoglobulin A: Strategic defense i n i t i a t i v e at the mucosal surface. Ann. Rev. Immunol., 4: 389-417.
37
THE STRUCTUREOF BOVINE SECRETORYCOMPONENT
D. BEALE Agricultural and Food Research Council, I n s t i t u t e of Animal Physiology and Genetic Research, Babraham, Cambridge, CB2 4AT, U.K.
ABSTRACT Beale, D., 1987. The structure of bovine secretory component. Vet. Immunol. Immunopathol., 17: 37-49. Bovine secretory component (SC) has been cleaved with trypsin into a series of fragments and their N-terminal amino acid sequences have been determined. The close homology with the known sequence of human SC has enabled the sequential order of the fragments to be deduced. The results indicate that bovine SC cQnsists of a single glycosylated polypeptide chain (Mr 74,000) folded into five globular immunoglobulin-like domains. A protein (Mr 94,000) has been isolated from detergent solubilised bovine epithelial membranes from l i v e r , intestine and mammary gland. This membrane protein is specific for the binding of J-chain linked IgM and IgA dimers. I t can be proteolytically cleaved into a water soluble SC-like portion and a detergent soluble hydrophobic portion. Bovine SC is therefore most l i k e l y to be the extracellular part of an e p i t h e l i a l receptor which mediates the transport of IgA dimers to mucosal surfaces. The various t r y p t i c fragments from bovine SC have been shown to d i f f e r in t h e i r relative binding a f f i n i t i e s for IgM and IgA dimers. The results imply that the f i r s t three domains of bovine SC are most involved in binding and domains 4 and 5 play subsidiary roles. Computerized prediction and modelling methods have been used to deduce possible t e r t i a r y and quaternary structures for SC. There are good indications t h a t the molecule has an elonaged "zig-zag" s t r u c t u r e s t a b i l i z e d by longitudinal inter-domain contacts. A model of SC bound to IgA dimer is presented. INTRODUCTION During a study of immune globulins in bovine milk Groves and Gordon (1967) isolated a previously undetected protein which they called glycoprotein-a.
It
was subsequently identified by Butler (1971) as bovine secretory component (SC) which had been reported by Mach et al. (1969) and Porter and Noakes (1970) to be present in bovine external secretions both in the free Form and as a component of SIgA. Physicochemical studies on bovine SC (Butler, 1971; Labib et a l . ,
1976) established i t
as a rather elongated molecule consisting of a
single glycosylated polypeptide chain of Mr 70-79,000.
One mole of SC was
found per mole of SIgA. A series of reports from a number of laboratories working with human and rabbit SC gradually established that the protein formed, or was related to, an 0165-2427/87/$03.50
© 1987 Elsevier Science Publishers B.V.
38 epithelial
receptor which mediates the t r a n s c e l l u l a r transport
of J-chain-
linked IgM and IgA polymers to mucosal surfaces (Brandtzaeg, 1985; Underdown and S c h i f f , 1986). My laboratory began to study bovine SC in 1984 with the i n i t i a l
aims of ( i )
locating the binding s i t e , (2) establishing the presence of a bovine epithelial polyimmunoglobulin receptor and (3) determining the primary structure and deducing secondary, t e r t i a r y and quaternary structures.
Progress towards the
f i r s t objective was made by preparing a t r y p t i c Fragment, of Mr 25,000, which binds polymeric immunoglobulin (Beale and Hopley, 1985) and locating i t s position in the SC molecule as described here.
The second aim was achieved by
isolating a protein from detergent solubilized epithelial membranes which bound polymeric immunoglobulin and had a structure similar to that of bovine SC (Beale and Hopley, 1986). The third aim was greatly helped when Mostov et al. (1984) published the amino acid sequence for the rabbit polyimmunoglobulin receptor, translated from a cDNA sequence, and E i f f e r t et al. (1984) published the sequence of human SC obtained directly from the protein.
These sequences
indicated that SC and the extracellular part of the receptor have five homology regions related to
immunoglobulin
domains.
We carried out computerized
secondary structure predictions on these sequences, deduced possibly t e r t i a r y structures and tested the v a l i d i t y of these by locating accessible and inaccessible sites for t r y p t i c cleavaje in bovine SC (Beale and Coadwell, 1986; and this paper). MATERIALS AND METHODS Preparation of bovine SC Milk
whey p r o t e i n s
were
fractionated
to
obtain
SC by ion-exchange
chromatography and gel F i l t r a t i o n as described by Butler (1971) and Labib et al. (1976). Preparation of bovine epithelial pol~immunoglobulin receptor The method of Kuhn and Kraehenbuhl (1979, 1981) as modified by Beale and Hopley (1986) was followed. Epithelial membranes from bovine l i v e r , intestine and mammary gland was solubilized in detergent and subjected to a f f i n i t y chromatography with rabbit IgG-Sepharose, rabbit
IgG - (anti-bovine SC)-
Sepharose and human IgM-Sepharose. Fragmentation with trypsin Solutions of protein (0.5%) in 0.15M NaCl, O.1M Tris-HCI, pH 8.0, were digested with trypsin (protein/enzyme ratio 100:1) For various times at 37oc. Then trypsin inhibitor at twice the weight of trypsin was added and the samples stored at -20oc.
39
High performance l i q u i d chromatography An LKB TSK G3OOOSWUltropac gel f i l t r a t i o n column (7.5 x 600 mm) was used in l i n e with an LKB 2150 HPLC pump, a Rheodyne 7125 injector, a Pye Unicam PU4020 UV detector and a Philips PM 8252 recorder.
Elution was carried out with 60mM
phosphate buffer, pH 7.4, in most cases, and the eluate monitored by absorption at 280 nm. SDS pol~acr~lamide gel electrophoresis The method of Laemmli (1970) as modified by Richardson and Feinstein (1978) was employed.
10% and 12% w/v polyacrylamide gels with 3% w/v stacking gels
were used with a discontinuous T r i s - g l y c i n e buffer containing 0.1% SDS. Samples were boiled in 1% SDS with or without mercaptoethanol.
Protein was
detected with Coomassie B r i l l i a n t Blue or by the s i l v e r stain method of Oshawa and Ebata (1983).
Stained gels were scanned d e n s i t o m e t r i c a l l y .
Protein
labelled with 1251 was detected by autoradiogrphy or by s l i c i n g and counting gels. Relative binding a f f i n i t ~ This was measured using the i n h i b i t i o n method of Socken and Underdown (1978) as modified by Beale and Hopley (1985).
Serial dilutions of the test protein
were mixed with a standard amount of 125 I-SC and the amount of radioactivity bound to a standard amount of IgM-Sepharose under standard conditions was measured. N-terminal amino acid sequencing This was carried out on an Applied Biosystems 470A Gas Phase Protein Sequencer in series with an Applied Biosystems 120A PTH Analyzer. RESULTS Fragmentation of free bovine SC Figure la shows the results of running reduced aliquots of t r y p t i c digests of bovine SC on SDS polyacrylamide gel electrophoresis. as densitometric scanning profiles.
They are represented
I t w i l l be seen that as digestion time
lengthens the main products occupy lower positions on the gels thereby i n d i c a t i n g lower molecular weights. themselves in groups on the gels.
Also the products tend to position
Fragmen%s Q and R, present at an early stage
of the digestion, run near the position of an Fdu' marker which consists of three immunoglobulin domains, two of them glycosylated.
Fragments S and T,
which appear at an early stage, and fragment A, which appears at a later stage, a l l run near the position of an Fdu marker which consists of two immunoglobulin domains, one of them glycosylated.
Fragments B and C, which appear at a late
40
-Fd~
R a
s
-Fdju
>
1
-p2m o
2
5
10
15
25
40
60 min
b
Figure 1.
Densitometric scanning p r o f i l e s from SDS polyacrylamide gel electrophoresis of a) reduced and b) unreduced aliquots of 0-60 min t r y p t i c digests of bovine SC. The positions of Fd'u, Fdu and B2M markers are indicated on the right.
stage of the digestion, run near to the position of B2-micro-globulin which consists of one immunoglobulin-like domain. Figure lb shows the results of running similar aliquots unreduced on SDS polyacrylamide gel electrophoresis.
It
is obvious that fragments Q, S and T
are now absent but there is more material in the position of SC.
This suggests
that fragments Q, S and T are only freely released by reduction. The interpretation of the fragmentation results is that bovine SC consists of f i v e globular immunoglobulin-like domains in accordance with the published sequence information
For human SC ( E i f f e r t
et
al.,
1984) and r a b b i t
polyimmunoglobulin receptor (Mostov et a l . , 1984). I n i t i a l t r y p t i c cleavage of SC produces a fragment Q, of three domain size, disulphide bridge to fragment S or T, of two domain size. degree of glycosylation.
Fragments S and T probably only d i f f e r in t h e i r Cleavage near to the disulphide bridge then freely
releases fragment R, of three domain size but s l i g h t l y smaller than fragment Q. Fragment R eventually gives rise to the two-domain fragment A.
Fragments B and
C are derived by further cleavage of both fragments S and T. Figure 2a shows the 280 nm absorption profiles from the HPLC of t r y p t i c digests of bovine SC.
Figure 2b shows the results of running reduced aliquots
41
elution .-.z,
SC
20min
A
C
peak SC
0
R
A
c
B
-68,ooo -43,000
-23,000
Figure 2.
a) 280 nm absorption profiles from gel f i l t r a t i o n HPLC of tryptic digests of bovine SC. b) Densitometric scanning profiles from SDS polyacrylamide gel electrophoresis of reduced aliquots of HPLC peaks shown in a).
of the HPLC peaks on SDS polyacrylamide
gel
electrophoresis.
represented as densitometric scanning p r o f i l e s .
They are
I t was found that peak R from
the I0 min digest consisted almost e n t i r e l y of fragment R of Mr 42,000.
Peaks
A, B and C from the 35 min digest contained single components (fragments A, B and C) of Mr 25,000, 14,000 and 12,000 respectively. N-terminal amino acid sequences of t r ~ p t i c fraQments These results are shown in Figure 3 where they homologous
sequences from human SC and rabbit
are
aligned
Ig receptor.
with
the
Computerized
sequence searching showed that these are the only s i g n i f i c a n t alignments. It
will
be seen t h a t
the
N-terminal
sequence of
i d e n t i c a l with the N-terminal sequence of the f i r s t
fragment R is
almost
homology region of human SC
42 bovine SC, fragment Q, bovine SC. fragraent R, human SC, ]st homology region, rabbit receptor, ]st honmlogy region,
N-terminus. N-terminus. N-terminus. N-terminus.
SPIFGPEEVD SPIFGPEEVDSVEGSSVSITC KSPIFGPEEVNSVEGNSVSITC PSSIFGPGEVNVLEGDSVSITC
bovine SC, fragment A, N-terminus. hu~an SC, Ist homology region, C-terminus. rabbit receptor, Ist homlogy region, C-terminus.
ELNFQVSLEVSQ RGLSFDVSLEVSQ RGLDFGVNVLVSQ
bovine SC, fragment B, N-terminus. human SC, 4th homology region, mlddTepert. rabbit receptor, 4th homOlogy region, mlddlepart.
LALLTVPGPVNYKVILNQLTDQDTGFYWC RLSLLEEPGNGTFTVILNQLTSRDAGFYWC RLALFEEPGNGTFSVVLNQLTAEDEGFYWC
bovine SO, fragment C, N-termlnus. ,,uman SO, 5th homo]ogy region, N-termlnus. rabbit receptor, 5th homology region, N-terminus.
VPLGEFSLKVPKVVDAWLSEPLKLSCYFPPKKYWFF KIIEGEPNLKVPGNVTAVLG£TLKVPCHFPCKFSSY£ QIVDGEPSPTIDK-FTAVQGEPVEITCHFPCKYFSSE
Figure 3.
and d i f f e r s
N-terminal amino acid sequences of t r y p t i c fragments from bovine SC compared with homologous sections from human SC ( E i f f e r t et a l . , 1984) and rabbit receptor (Mostov e t a l . , 1984).
at only one position from the N-terminal
sequence of bovine SC
published by Labib et a l . (1976). The N-terminal sequence of fragment A is very homologous with the C-terminal sequences of the f i r s t
homology regions of human SC and rabbit receptor.
The
homologous sections are immediately preceded by arginine residues which would provide poteqtial sites for t r y p t i c cleavage. The N-terminal
sequence of
fragment 3 is
very homologous with the middle
parts of the fourth homology regions of human SC and rabbit receptor. each case the
homologous
section
is
immediately preceded
Again in
by an arginine
residue. The N-terminal amino acid sequence of fragment C is very homologous with the N-terminus of the f i f t h is
homology region of human SC where the relevant section
immediately preceded by a l y s i n e residue which would provide a potential
s i t e for t r y p t i c cleavage. The fragmentation process The results
of t r y p t i c
fragmentation and N-terminal sequence analysis are
summarized d i a g r a m m a t i c a l l y
in
Figure
4.
The f i v e
domains
of
SC are
represented as loops, each containing an intra-domain disulphide bridge as in immunoglobulin domains.
Initial
tryptic
cleavage occurs w i t h i n domain 4 and
gives rise to fragment Q disulphide bridge to fragment T by the intra-domain bridge of domain 4.
Fragment Q consists of domains I ,
terminal portion of domain 7. domain 4 plus most i f
not a l l
2 and 3 plus an N-
Fragment T consists of a C-terminal portion of of domain 5.
Further cleavage near the N-
terminus of domain 4 then releases fragment R which consists of domains 1, 2
43 R
NH2
I COOH
Q
Figure 4.
and 3.
T
-
-
Diagrammatic representation of the t r y p t i c fragmentation of bovine SC. The SC polypeptide chain is shown as consisting of f i v e domains, represented as loops, each containing an intra-domain disulphide bridge.
Cleavage towards the C-terminus of domain 1 then gives rise to fragment
A which consists of domains 2 and 3 plus a portion of the C-terminal end of domain 1.
Further cleavage within domain 4 and at i t s C-terminus gives rise to
fragment B, which consists of a C-terminal section of dQmain 4 plus most i f not all of domain 5, and C, which consists of domain 5. Relative binding a f f i n i t i e s of t r y p t i c fragments The inhibition results are shown graphically in Figure 5. fragment
I t is clear that R (domains 1, 2 and 3) inhibits the binding of 1251-SC to IgM-
7~
.
.
.
3O
,
2
|
4
,
, ,
6
, ,
8 10
30
60
100
po
Figure 5.
Inhibition of 1251-SC binding to IgM-Sepharose. • R, ~ fragment A, A Fragment B, ~ Fragment C.
SC, V fragment
44 Sepharose to a similar extent as bovine SC does. Fragment A (domains 2 and 3) also i n h i b i t s
but
is
considerably less effective than SC or fragment R.
Fragment B (part of domain 4 plus domain 5) and fragment C (domain 5) do not inhibit. These results imply that SC domains I and 2 are most involved in binding polymeric immunoglobulin, domain 3 is also l i k e l y to be involved but domains 4 and 5 probably play subsidiary roles. Fragmentation of bound SC Figure 6 shows the results of digesting SIgA (bovine SC bound in vivo to IgA dimers) and 1251-SC bound
in v i t r o to
IgM and IgA dimers.
scanning profiles and radioactivity profiles are shown.
It
Densitometric will
be seen
(Figure 6a,b) that even after 2 hr digestion of SIgA, fragment ~ is s t i l l present in good yield whereas only a 1 hr digestion of unbound SC degrades i t to Fragments A, B and C (Figure 6c,d).
Similar results were obtained when
1251-SC was bound in v i t r o to IgM or IgA dimers (Figure 6e-h) except that some Fragment R was present as well as Fragment Q. be released from digested SIgA by reduction.
Further, Fragment Q could only I t has the same N-terminal amino
acid sequence as Fragment R (Figure 3). These results imply that during the binding of polymeric immunoglobulin in vivo the potential t r y p t i c cleavage sites near the C-terminus of domain 1 an the N-terminus oF domain I become burised and a disulphide bridge Forms between IgA and domain I , 2 or 3. v i t r o but there is l i t t l e
a b SC
Figure 6.
A very similar burying of cleavage sites occurs in i f any disulphide bridging.
C
H
sI
f
g
h
SCl I
Fragmentation of bovine SC bound in vivo and in v i t r o . Unfilled profiles represent densitometric scans of SDS polyacrylamide gel electrophoresis of reduced samples. Filled profiles represent radioactivity of gel slices and are shown on the r i g h t of the respective densitometric scan. SIgA, a) undiqested and b) digested 2h. SC, c) undigested and d) digested lh. 1251-SC bound in v i t r o to IgM, e) undigested and f) digested 2 h. 1251-SC bound in v i t r o to IgA dimers, g) undigested and h) digested 2h.
45
~'
Figure 7.
NH 2
Diagra~natic representation of the immunoglobu!in-like SC domain. B-strands are shown as long arrows. Connecting sequences between strands are shown as l i n e s . The four shaded B-strands make ;~p one B-shee and the f i v e ,Jnshaded B-strands :hake up the other B-sheet. SC domains 1-4 have t e r t i a r y structures similar to that shown but domain 5 has one B-strand less in each sheet. Short arrows indicate potential sites for t r y p t i c hydrolysis which are cleaved in one or other of the bDvine SC domains. Black diamonds indicated potential sites for t r y p t i c hydrolysis which are not cleaved in '~n~'~qe SC.
Accessible and inaccessible sites for tr~ptic hydrol~sis in the t e r t i a r ~ structural models of SC domains Figure 7 is a diagra,mnatical representation of the t e r t i a r y structures of SC domains 1-4 computed in my laboratory (Beale and Coadwell, 1986).
They have
many of the characteristics of immunoglobulin domains and consist of a f i v e stranded B-sheet and a four-stranded B-sheet joined by an intra-domain disulphide bridge. The t e r t i a r y structure of domain 5 is very similar but has two less B-strands. It
will
be seen that
potential
actually cleaved in bovine SC al] positions.
sites lie
for t r y p t i c hydrolysis that are
outside the B-strands at exposed
In contrast, the potential sites For t r y p t i c hydrolysis that are
not cleaved in bovine SC a l l positions.
lie
w i t h i n the B-strands
at inaccessible
Fragmentation of bovine epithelial pol~immunoglobulin receptor In these experiments a mixture of 1251-receptor and unlabelled SC was digested with trypsin in 0.1% detergent and reduced aliquots were run on SDS polyacrylamide gel electrophoresis. The results are shown as densitometric scans and radioactivity profiles in Figure 8.
46 a
p
04,00074,000-
P
m
Figure 8.
Fragmentation of a mixture of bovine SC and 1251 labelled bovine epithelial polyimmunoglobulin receptor. Unfilled profiles represent densitometric scanning p r o f i l e s from SDS polyacrylamide gel electrophoresis of reduced samples. F i l l e d p r o f i l e s represent r a d i o a c t i v i t y of gel slices and are shown on the right oF the respective densitometric scans, a) undigested mixture, b) 10 min digest, c) 25 min digest, d) 40 min digest. Molecular weights are shown on l e f t ,
a
b
iD
Figure 9.
Phase separation with Triton X-114 of bovine e p i t h e l i a l receptor and t r y p t i c fragments. The profiles represent r a d i o a c t i v i t y of SDS polyacrylamide gel slices of reduced samples, a) undigested sample, detergent phase on l e f t and aqueous phase on right, b) sample digested 20 min, detergent phase on l e f t and aqueous phase on right.
In the undigested mixture (Figure 8a)most of the r a d i o a c t i v i t y was detected at a position indicative of Mr 94,000 although appeared at Mr 74,000 coincident with SC.
a significant
amount also
As digestion proceeded (Figure 8b-d)
most of the r a d i o a c t i v i t y occupied positions of decreasing molecular weight that were exactly coincident with those of SC fragments. When the experiments were repeated using phase separation in Triton X114 (Bordier, 1981) the r a d i o a c t i v i t y in the undigested mixture was found mainly in the detergent phase (Figure 9a) but after t r y p t i c digestion of the mixture nearly all the r a d i o a c t i v i t y appeared in the aqueous phase (Figure 9b).
47 DISCUSSION The experiments described in this paper provide good evidence that bovine SC is derived from an epithelial membrane receptor which mediates the transport of IgM and IgA polymers across the epithelial layer to the mucosal surface.
The
extracellular part of the detergent solubilized receptor is easily identical with SC.
The presence of SC in SIgA is presumably an aftermath of the
transport process. The membrane protein appears to be the bovine equivalent of the human polymeric immunoglobulin receptor reported by Mostov and Blobel (1982) and the rabbit polymeric immunoglobulin receptor described by Kuhn and Kraehenbuhl (1981) and Mostov et al. (1984). The bovine epithelial receptor and SC can be cleaved by trypsin into a series of well characterized fragments whose locations in the SC molecule have been determined by N-terminal sequence analysis.
Fragments obtained from human
SC by Kobayashi et al. (1973) but not f u l l y characterized are probably the human equivalents of the bovine SC fragments. The physicochemical properties of the t r y p t i c fragments support the concept of a five domain SC molecule put forward by Mostov et al. (1984) and E i f f e r t et al. (1984) based on internal sequence homologies. The fragments have different binding a f f i n i t i e s which suggest that the f i r s t three domains of SC are most involved in binding polymeric immunglobulin.
The r e l a t i v e proteolytic
resistance of the f i r s t three domains when SC is complexed with IgM and IgA polymers indicates that there are very close contacts between receptor and ligand (Beale, 1985). The locations of accessible and inaccessible sites for t r y p t i c hydrolysis in the bovine SC molecule support putative t e r t i a r y structures for the SC domains (Beale and Coadwell, 1986) which are very immunoglobulin-like as predicted by Mostov et al. (1984) and E i f f e r t et al. (1984). However, possibly quaternary structures derived by assembling the domains do not allow close inter-domain contact as found in immunoglobulin molecules. Experimental support is provided by the fact that we could not detect any significant interactions between the various Fragments of bovine SC. The main interdomain contacts which determine the quaternary structure are therefore l i k e l y to be weak longitudinal ones. T h i s leads to two quaternary structural models.
One has a horseshoe shape but would need an inter-domain
disulphide bridge between domains 1 and 5 to s t a b i l i z e the structure. I n i t i a l l y we thought that such a bridge might exist (Beale and Hopley, 1985) but have been unable to substantiate this experimentally. The alternative model which we now favour has a zig-zag shape which does not require a disulphide bridge and a similar structure for human SC has been proposed by Pumphrey (1986). A diagrammatical representation of the bovine SC molecule is
48
i
a
b
c
Figure 10. a) Putative quaternary s t r u c t u r e f o r bovine SC. The f i v e immunoglobulin-like domains are represented as c y l i n d e r s which interact l o n g i t u d i n a l l y to form a zig-zag structures, b) A similar representation of the Fc regions of two IgA or IgM monomers linked by J-chain which is shown as an oblong box. c) Diagrammatic representation of bound SC. Domain 1 is interacting with a CH2 domains of one of the Fc monomers and probably forms a disulphide bridge (not shown). Domain 3 is interacting with the CH3 domain of the other Fc monomer but would not Form a disulphide bridge. Domain 2 is interacting with J-chain. d) SC bound to an a l t e r n a t i v e arrangement of the Fc regions of two IgA monomers. The interactions are similar to those in c).
given in Figure 10 and the binding of J-chain-linked IgA dimers represented.
is
also
The domains are i l l u s t r a t e d as cylinders following the convention
of Schiffer et al. (1973) for immunoglobulin domains. Within each cylinder the peptide chain is folded into two B-pleated sheets linked by the intra-domain disulphide bridge.
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49
derived by primary structure comparisons with immunoglobulins. Comp. Biochem. Physiol. (in press). Beale, D. and Hopley, J.G., 1985. Fragmentation and reduction of bovine secretory component. Preparation of a biologically active fragment and some evidence for a multiple-domain structure. Biochem. J., 226: 661-667. Beale, D. and Hopley, J.G., 1986. A bovine epithelial membrane protein that binds polymeric inmunoglobulin and has a structure related to that of bovine secretory component. Biochem. J., 233: 37-40. Bordier, C., 1981. Phase separation of integral membrane proteins in Triton X114. J. Biol. Chem., 256: 1604-1607. Brandtzaeg, P., 1985. Role of J chain and secretory component in receptormediated glandular and hepatic transport of immunoglobulins in man. Scand. J. Immunol., 22: 111-146. Butler, J.E., 1971. Physicochemical and immunochemical studies of bovine IgA and glycoprotein-a. Biochim. Biophys. Acta., 251: 435-449. Eiffert, H., Quentin, E., Decker, J., H i l l m e i r , S., Hufschmidt, M., Klingmuller, 0., Weber, M.H.. and Hilschmann, N., 1984. The primary s t r u c t u r e of human free secretory component and the arrangement of disullphide bonds. Hoppe-Seyler's Z. Physiol. Chem., 365: 1489-1495. Groves, M.L. and Gordon, W.G., 1967. Isolation of a new glycoprotein-a and a globulin From individual cow milks. Biochemistry, 6: 2388-2394. Kobayashi, K., Yaerman, J.P. and Hereman, J.F., 1973. Studies on human secretory IgA ( I I ) . Comparative studies on a fragment of secretory component derived from secretory IgA and fragments obtained by enzymatic digestion of free secretory component. Immunochemistry, I0: 73-80. Kuhn, L. and Kraehenbuhl, J.P., 1979. Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells. J. Biol. Chem., 254: 11072-11081. Kuhn, L. and Kraehenbuhl, J.P., 19981. The membrane receptor for polymeric immunoglobulin is structurally related to secretory component. Isolation of membrane secretory component frown rabbit liver and mafmnary gland. J. Biol. Chem., 256: 12490-12495. Labib, R.S., Calvanico, N.J. and Tomasi, T.B., 1976. Bovine secretory component. Isolation, molecular size and shape, composition, and NH2terminal amino acid sequence. J. Biol. Chem,, 251: 1969-1974. Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Mach, J.P., Pahud, J.J. and Is]iker, H., 1969. IgA with secretory piece in bovine colostrum and saliva. Nature, 223: 952-954. Mostov, K.E. and Blobel, G.A., 1982. A transmembrane precursor of secretory component. The receptor for t r a n s c e l l u l a r transport of polymeric immunoglobulins. J. Biol. Chem., 157: 11816-11821. Mostov, K.E., Friedlander, M. and Blobel, G., 1984. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulinlike domains. Nature, 308: 37-43. Ohsawa, K. and Ebata, N., 1983. Silver stain for detecting lO-femtogram quantities of protein after polyacrylamide gel electrophoresis. Analyt. Biochem., 135: 409-415. Porter, P. and Noakes, D.E., 1970. Immunoglobulin IgA in bovine serum and external secretions. Biochim. Biophys. Acta, 214: 107-116. Pumphrey, R.S.H., 1986. Computer models of the human itnmunoglobulins: binding sites and molecular interactions. Immunology Today, 7: 206-211. Richardson, N.E. and Feinstein, A., 1978. Mouse i n t r a c e l ] u l a r IgM. Structure and identification of a free thiol group. Biochem. J., 175: 959-967. Socken, D.J. and Underdown, B.J., 1978. Comparison of human, bovine and rabbit secretory component-immunoglobulin interactions. Immunochemistry, 15: 499506. Schiffer, M., Girling, R.L., Ely, K.R. and Edmundson, A.B., 1973. Structure of a -type Bence Jones protein at 3.5A resolution. Biochemistry, 23: 46204631. Underdown, B.J. and Schiff, J.M., 1986. Immunoglobulin A: Strategic defense i n i t i a t i v e at the mucosal surface. Ann. Rev. Immunol., 4: 389-417.