Planta (1990)182:64~71

P l ~ n t a 9 Springer-Verlag1990

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12 J.L. Jones*, J.A. Callow, and J.R. Green* *

School of Biological Sciences, University of Birmingham, P.O. Box 363, Birmingham BI5 2TT, UK Received 2 November 1989; accepted 5 May 1990 Abstract. Sperm o f the brown alga F u c u s s e r r a t u s are

highly differentiated, biflage!late, naked cells. Immunolocalisation studies, employing monoclonal antibodies (MAbs designated FS1 to FS12) raised against antigens of these sperm cells, have revealed that some sperm surface components are distributed over the entire cell, whereas others are restricted to, or occur preferentially on, the surface of the anterior flagellum or cell body. This report describes the use of these MAbs in Westernblot procedures and antigen-modification binding assays to determine the nature of these sperm surface components. Monoclonal antibodies which bind to antigens found on the cell body and both flagella (FS3, FS4, FS6, FS8, FS10) recognise carbohydrate epitopes of a high-molecular-weight glycoprotein ( M r = 2 0 5 kDa). These MAbs were initially chosen at random from a much larger number of antibodies which bound to sperm in a similar fashion, indicating that this glycoprotein is an immunodominant antigen. Though these MAbs compete under conditions of limited antigen availability, differences in the effects of periodate on antibody binding and differences in other binding data indicate that the MAbs recognise epitopes of this glycoprotein which are neighbouring or overlapping, rather than common. The MAb FS9, which has a similar binding pattern to the above antibodies, also seems to bind to carbohydrate epitopes, but the antigen recognised by this antibody could not be identified in Western-blotting procedures. The MAbs FS7 and FS12, which bind to the mastigonemes on the anterior flagellum and to the cell body and posterior flagellum, recognise a set of glycoproteins in * Present address: Department of Chemistry and Biochemistry, Campden Food and Drink Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK ** To whom correspondence should be addressed Abbreviations: ELISA=enzyme linked immunosorbent assay; HRP-RAMIG = horseradish-peroxidase-labelled rabbit anti mouse immunoglobulin; Ig = immunoglobulin; kDa = kilodalton; MAb = monoclonal antibody; Mr=relative molecular mass; PBS=phosphate-buffered saline; SDS-PAGE=sodium dodecyl sulphate polyacrylamide gel electrophoresis

the molecular-weight range 40-250 kDa. The evidence indicates that the antibodies are binding to N-linked carbohydrate side chains of these glycoproteins. Three MAbs that bind to the anterior flagellum (FS2, FS5 and F S l l ) recognise protein antigens in the molecularweight range 90-250 kDa; it is not known whether these antigens are glycosylated. The MAb FS1, which binds primarily to the sperm cell body, could not be used in enzyme-linked immunosorbent assays or Western-blotting procedures and the antigen recognised by this antibody is so far uncharacterised. Key words: Cell surface - Fertilisation (recognition) Fucus -

Glycoprotein - Sperm (surface antigens)


Sperm of the brown alga F u c u s s e r r a t u s are highly differentiated cells. Immuno-localisation studies with a panel of twelve monoclonal antibodies (MAbs, designated FSI-FS12), have shown that some surface antigens are restricted to particular regions of the sperm whereas others are distributed over the entire plasma membrane (Jones et al. 1988). These distributional studies are summarised in Table 1. Several MAbs bind to the entire sperm including both flagella (FS3, FS4, FS6, FS8, FS9 and FS10) and the labelling of the antibodies is concentrated on the body. Two MAbs (FS7 and FS12) also bind to the entire sperm but their binding is highly concentrated on the anterior region of the cell. This is due to intense binding of these antibodies to the mastigonemes on the anterior flagellum, and this is in contrast to the MAbs of the previous group which label the shaft of the anterior flagellum. Other MAbs show more restricted binding to the sperm surface. FS1 binds primarily to the sperm body: it also labels the mastigonemes on the anterior flagellum very weakly. Three MAbs (FS2, FS5 and F S l l ) bind preferentially to the anterior

J.L. Jones et al. : Fucus sperm surface antigens


Table l. Binding patterns of MAbs FS1-FS12 to Fucus serratus sperm and eggs. Binding of MAbs to sperm was determined by indirect immunofluorescence and immunogold labelling. The intensity of binding was recorded as: - absent; _ very weak; + weak; + + intermediate; + + § strong. Some MAbs bound the mastigonemes (M) but the others bound instead to the shaft of the anterior flagellum. Binding of MAbs to egg vesicles was determined by ELISA MAb

FS4(IgM) FS3(IgM) FS6(IgG1) FS8(IgM) FS9(IgG2b) FS10(IgM) FSI(IgM) FSV(IgG1) FS12(IgG1) FS5(IgM) FS2(IgG1) FS1 l(IgM)



Anterior flagellum


Posterior flagellum

+ + + + + + + _+(M) + + +(M) + +(M) + + + (M) + + (M) + +

+ + + + + + + + + + + + + + + + + + + + _+ +_ -

+ + + + + + + -+ + --

+ + ---+ + -

f l a g e l l u m : F S 1 1 b i n d s to t h e p l a s m a m e m b r a n e o f this flagellum, while FS2 and FS5 bind to the mastigonemes, t h o u g h t h e y a l s o label t h e s p e r m b o d y v e r y w e a k l y . These findings can be compared with studies of animal ( m a m m a l , sea u r c h i n ) s p e r m s u r f a c e s w h e r e s u c h r e g i o n al h e t e r o g e n e i t y h a s a l s o b e e n o b s e r v e d , d i f f e r e n t r e g i o n s e a c h f u l f i l l i n g specific r o l e s d u r i n g f e r t i l i s a t i o n ( S a l i n g e t a l . 1985; S a l i n g a n d L a k o s k i 1985; T r i m m e r e t a l . 1985; P r i m a k o f f et al. 1987). F o u r o f t h e M A b s ( F S 2 , FS4, FS5 and FS9) cross react with egg membranes and F S 1 0 b i n d s s p e r m in a s p e c i e s - p r e f e r e n t i a l m a n n e r . In the present paper we have used Western blotting a n d a n t i g e n - m o d i f i c a t i o n t e c h n i q u e s to c h a r a c t e r i s e t h e antigens recognised by the panel of MAbs.

Materials and methods Plant material and sperm-lysate preparation. Gametes of Fucus serratus were prepared as described previously (Jones et al. 1988). Suspensions of sperm cells were centrifuged at 1000.g for 5 min, resuspended in sea water (10 s cells-ml-1) and diluted with an equal volume of 1% (v/v) Triton X-100 (Sigma, Poole, Dorset, UK). Following a 30-min incubation at 4 ~ C, the mixture was centrifuged at l l 6 0 0 - g for 10 min to pellet debris and the supernatant harvested. Phenylmethylsulfonyl fluoride (100 mM in isopropanol; Sigma) was added to a final concentration of 5 m M and the lysate was stored at - 20~ C. Protein content of the lysate was determined using the Bio-Rad Bradford protein assay, performed according to the manufacturers instructions (Bio-Rad, Watford, Hefts., UK). Monoclonal antibodies. The MAbs used in this study, designated FS1-FS12, are those produced and described by Jones et al. (1988). Their binding characteristics are summarised in Table 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blotting. This was carried out using the BioRad minigel/transblot apparatus in accordance with the manufac-

turers instructions and modifications of the methods of Laemmli (1970) and Horst-Walker and Koepsell (1987) as below. To 400 p.1 of sperm lysate (350/.tg protein.m1-1) was added 800 gl of 10% (w/v) trichloracetic acid and the mixture incubated on ice for 5 min. The precipitated protein was pelleted by centrifugation (11600-g for 10 rain), the pellet was washed once with acetone and finally resuspended in 100 gl reducing sample buffer (0.125 M Tris-HC1, pH 6.8; 20% (v/v) glycerol; 5% (w/v) SDS; 5% (v/v) 2-/3-mercaptoethanol; 0.0005% (w/v) bromophenol blue) and incubated at 37~ for 15 min. Aggregated material was removed by a final centrifugation step (11600.g for 30 s) and 50 gl was loaded onto each of two 10% minigels, which were run concurrently at 100 V for approx. 1 h at 20 ~ C. After transfer of the separated proteins onto nitrocellulose (Hybond C; Amersham International, Amersham, Bucks., UK) with the transblot apparatus (Towbin et al. 1979), the blotted membrane was then incubated in phosphatebuffered saline (PBS) containing 0.1% (w/v) sodium azide for 16 h at 30~ for renaturation of the blotted proteins (Horst-Walker and Koepsell 1987). Thereafter, to check protein transfer to the nitrocellulose, the edges of the membrane were removed and stained for 5 min at 20~ in 10% (v/v) amido black (Sigma) in 45% (v/v) methanol and 7% (v/v) acetic acid (BDH Chemicals, Poole, Dorset, UK) in water, and subsequently destained in 90% (v/v) methanol, 2% (v/v) acetic acid in water. The remaining area of the blot was cut into strips for probing with MAbs. The strips were incubated with constant agitation, first in PBS containing 10% (v/v) newborn calf serum (NCS; Gibco, Uxbridge, Middx., UK) and 0.5% (v/v) Tween 20 (Sigma), for 4 h at 20 ~ C, and then in MAb supernatant diluted 1:2 with PBS containing 10% (v/v) NCS, 0.5% (v/v) Tween 20, 2 M D-glucose and 20% (v/v) glycerol, for 16 h at 4 ~ C. Following four washes, each of 10 min, with PBS containing 0.5% (v/v) Tween 20, the strips were incubated in horseradish-peroxidase-conjugated rabbit anti-mouse immunoglobulins ( H R P - R A M I G ; Dakopatts, High Wycombe, U K ) d i l u t e d 1/100 with PBS containing 10% (v/v) NCS and 0.5% (v/v) Tween 20 for 2 h at 20 ~ C. Following a further five washes (1 x 0.5% (v/v) Tween 20 in PBS; 1 x 0.05% (v/v) Tween 20 in PBS; 3 x PBS) strips were incubated, with agitation, in substrate solution (i volume of methanol containing 3 m g - m l 1 4-chloro-l-naphthol (Sigma), 5 volumes of PBS, hydrogen peroxide added to 0.01% (v/v) for 30 min at 20 ~ C in darkness, subsequently washed three times with water, dried and stored in darkness.

Enzyme-linked immunosorbent assay ( ELISA ). This was performed on Immulon I microtitre plates (Gibco) which were coated in sequence with each of the following: (i) 100 lal per well of poly-Llysine (10; Sigma) for 2 h ; (ii) 50 ~tl per well of sperm lysate (50 ~tg 1) for 2 h at room temperature; (iii) 250 per well of blocking solution containing 10 mg-ml-1 bovine serum albumin (BSA; Sigma) and 0.02% (w/v) sodium azide in PBS for more than 12 h at 4 ~ C. Between steps, plates were washed twice with PBS (200 ~tl per well) and stored with blocking solution at 4 ~ C. In the antigen-modification experiments, the adsorbed lysate was treated with the appropriate agent (see below) for the appropriate period, and washed at least three times prior to blocking the plate, i.e. just prior to step (iii). For testing binding of MAbs, wells received each of the following in sequence: (i) 50 ~tl of hybridoma culture supernatant for at least 2 h at 20 ~ C; (ii) 4 x 200 I-tlPBS, blotting dry between washes; (iii) 100 ~tl of alkalinephosphatase-conjugated rabbit anti-mouse immunoglobulin (APR A M I G ; Dakopatts) diluted 1/600 with 0.5 mg . m l - t BSA in PBS, for at least 2 h at 20 ~ C; (iv) 4 x 200-1xl washes as above; (v) 100 lal of 1 mg- m l - 1 p-nitrophenylphosphate (Sigma) freshly dissolved in 9.7% (v/v) diethanolamine (Sigma) buffer pH 9.8 for 1 h at 20 ~ C; (vi) 50 ~tl of 3 M NaOH was added to stop the reaction. Optical density was read at 405 nm. Antigen-modification procedures and assays. 1) Periodate oxidation: immobilised antigen (either on an ELISA plate or nitrocellulose) was washed once with 50 mM sodium acetate buffer (pH 4.5). Sodi-

J.L. Jones et al. : Fucus sperm surface antigens

66 um metaperiodate (20 mM NaIO4 in 50 mM sodium acetate buffer pH 4.5) was added to treated wells, while control wells received buffer alone. Following incubation for the appropriate period in darkness at 4~ C, wells were washed three times with acetate buffer and once with PBS before blocking. Reduction of the periodategenerated aldehyde sites with sodium borohydride (NaBH4 - 7 mg. ml- ~ for 2 h) was found to be without effect on the MAb-binding pattern and was not subsequently routinely performed. 2) Trypsin digestion : immobilised antigen was washed once with PBS, incubated in trypsin (BDH) solution (1 mg-m1-1 in PBS) at 37~ for the appropriate time, washed twice with PBS, incubated for 10 min in 0.1 trypsin inhibitor (Sigma) in PBS and then blocked. Controls received PBS without trypsin but were otherwise treated similarly. 3) Pronase digestion of immobilised antigen: following one wash with PBS, antigen received Pronase (Protease XIV; Sigma) at 0.1 unit per ~tg of lysate at 37~ C for the appropriate period, was washed four times with PBS and blocked. Controls received PBS without Pronase. 4) Pronase digestion of lysate suspension: Pronase conjugated to agarose beads (Sigma) was added to sperm lysate at 0.017 units per lag protein (125 lag protein in 350 lal lysate) and the mixture incubated, with frequent mixing, at 37~ C for 6 h. Protease beads were then pelleted by centrifugation at 11600.g for 10 min and the supernatant (digested lysate) coated on to polyL-lysine plates as described above. Control lysate was incubated without the beads but otherwise treated similarly. 5) Boiling: sperm lysate (protein at 350 -~) was boiled for 1 h, cooled and diluted to 50 with PBS. A control sample was incubated at 20~ C and diluted similarly. Each sample was used to coat half of the same ELISA plate, at 2.5 gg protein per well, and the ELISA performed as above. 6) Endo-F deglycosylation : immobilised lysate was treated with Endoglycosidase-F (endo-fl-N-acetylglucosaminidase F; EC; Boehringer, Mannhein, FRG), 0.1 Units. lag ~ protein, in 50 mM phosphate buffer (pH 7.1) for 24 h at 37~ and then washed three times with PBS. Controls received phosphate buffer alone. 7) Mild alkaline hydrolysis ( fl-elimination ) : immobilised lysate was washed once with PBS, incubated in 50 mM NaOH for 24 h at 20~ C, washed a further three times with PBS and then blocked. Controls received water in place of NaOH, but were washed with PBS.

Dot-blot assays. This was performed using the Bio-Rad immuno dot-blot apparatus. A nitrocellulose sheet (Hybond C; Amersham) was presoaked in distilled water and loaded into the dot-blot apparatus. Sperm lysate solution (20 lal of 5 lag-m1-1) was added to each well and drained under gravity (30 min) and excess solution drained by suction. The appropriate antigen-modifying reagent (e.g. periodate or trypsin) was added to the wells, the whole apparatus incubated accordingly and the wells finally drained by suction. Following washing, blocking solution (100 lal of 10 mg-ml-1 BSA in PBS) was added to the wells and drained (30 min gravity, then suction). Following a further wash with PBS the monoclonal antibody in the form of tissue-culture supernatant was added (50 lal per well), incubated for 30 min and then removed by suction. Following a further two washes, 50 gl of HRP-RAMIG (1/100 in 0.5 BSA in PBS) was added to each well, incubated for 30 min and excess removed by suction. After three PBS washes the nitrocellulose sheet was removed from the apparatus, rinsed in a tray of distilled water and allowed to develop in substrate solution (as used for Western blots above) for 10 min. The sheet was then rinsed in distilled water, dried and stored in an air-tight polythene bag in darkness. Competition assays. The additivity ELISA employed to determine whether the MAbs bind distinct epitopes is adapted from that of Friguet et al. (1983). It is based on the principle that, when mixed together, two MAbs which bind distinct epitopes produce an optical density in ELISA which is the sum of the two optical densities obtained when the MAbs are added separately. The optimal amount of sperm lysate used to coat plates for this assay was 0.1 lag protein per well for MAbs FS3, FS4, FS6, FS8, FS10, FS11,

and 0.5 lag per well for MAbs FS2, FS5, FS7 and FS12. Saturating levels of MAbs and AP-RAMIG were used in the assay. The MAbs (50 lal) were added to the wells in the appropriate pairwise combinations (see results) with the appropriate controls (undiluted MAb, MAb diluted 1:1, control MAb [to rat bone cells]). Binding of MAb was detected using AP-RAMIG diluted 1:400 and mixed with unlabelled RAMIG (Dakopatts) diluted 1:20 in PBS containing 0.5 1 BSA. The antibody incubation periods were increased to 3 h. Following addition of substrate the reaction was stopped after 40 min in the linear phase of the reaction. Additivity indices were calculated from the equation of Friguet et al. (1985) viz. : AI = {[2 x AI +2/(A1 + Az)] - 1} x 100 where A1 and A2 are the respective optical densities obtained when the two MAbs are used alone, and A1+2 is the optical density obtained when the two MAbs are added together.

Results Western blotting P r o t e i n precipitated from sperm lysate was subjected to S D S - P A G E , blotted o n to nitrocellulose a n d p r o b e d with M A b s . The g r o u p o f M A b s c o m p r i s i n g FS3, FS4, FS6, FS8 a n d FS10, which b i n d to the entire sperm surface, all b o u n d to a p r o t e i n which f o r m e d a b r o a d b a n d with a n a p p r o x i m a t e relative m o l e c u l a r mass (Mr) of 205 k D a (Fig. 1 a). The b a n d f o r m e d b y F S I 0 was consistently n a r r o w e r t h a n t h a t o b t a i n e d with the other M A b s . The M A b s FS7 a n d FS12, which recognise mastig o n e m e s a n d b i n d to a lesser extent to the rest o f the sperm, gave identical b a n d i n g patterns, f o r m i n g multiple b a n d s t h r o u g h o u t the Mr range 40-250 k D a (Fig. I b). FS2, FS5 a n d F S l l labelled a series o f b a n d s , which often gave the a p p e a r a n c e o f a single diffuse b a n d , over the Mr range 90-250 k D a . The b a n d i n g p a t t e r n s o f FS2 a n d FS5, which b o t h label m a s t i g o n e m e s o n the a n t e r i o r flagellum, were identical, whereas FS11 which labels the sheath o f the a n t e r i o r flagellum u s u a l l y labelled a n t i g e n s d o w n to a lower Mr. FS1 a n d FS9 did n o t label a n t i g e n in Western blots o f a n u m b e r of S D S - P A G E gels at 12%, 10%, 8.5% or 7.5% (data n o t shown).

Competition-binding assays Based o n the results o f the Western blots a n d o n previous d i s t r i b u t i o n a l studies (Table 1), three assays were perf o r m e d to d e t e r m i n e w h e t h e r M A b s with similar b i n d i n g characteristics recognise distinct epitopes. A n additivity index (AI) o f 5 0 - 1 0 0 % indicates that the two M A b s b i n d distinct epitopes whereas lower values indicate c o m p e t i t i o n is o c c u r r i n g between the two M A b s . F r o m Table 2 it can be seen that M A b s which exhibit similar d i s t r i b u t i o n a l b i n d i n g p a t t e r n s [(A) FS3, FS4, FS6, FS8 a n d F S 1 0 ; (B) FS2 a n d F S 5 ; (C) FS7 a n d F S I 2 ] tend to c o m p e t e with AIs lower t h a n 20%. FS11 was i n c l u d e d as a positive c o n t r o l in (A) to prove that additivity between n o n - c o m p e t i n g M A b s could be o b t a i n e d ; it does n o t c o m p e t e with M A b s FS3, FS4, FS6, FS8 or FS10,

J.L. Jones et al. : Fueus sperm surface antigens

67 Table 2A-C. Additivity indices of the various combinations of MAbs. Three groups of assays were performed, viz: A MAbs FS3, 4, 6, 8, 10 and including FSll as a positive control to prove that additivity is obtained for non-competing MAbs; B MAbs FS2 and FSS; C MAbs FS7 and FS12. Additivity indices were calculated using the equation: AI =([2{A1 +2}/[A1 +A2]}-- 1) x 100, where A1 and A2 are the respective optical densities when the two MAbs are used alone and A1 +2 is optical density of the combined MAbs (A) MAb








3.6 . . . . . .

13.7 2.1 . . .

0.9 2.6 3.9

--5.4 -- 1.6 --6.0 5.7

8.4 19.1 19.6 0.0 9.8

80.6 53.3 102.0 57.1 86.2 2.1


Fig. 1 a, b. Western blotting: Fucus serratus sperm lysate was subjected to SDS-PAGE (reducing conditions, 10% gel) with subsequent blotting to nitrocellulose and was probed with MAb, binding of which was detected using HRP-RAMIG followed by peroxide and 4-chloro-naphthol. Controls ((7) involved using a MAb raised to rat bone cells, a MAbs FS3, FS4, FS6, FS8, FS10 and control. b MAbs FS7, FS12, FS2, FS5, FSll and control

thereby validating the assay. FS9, though having a similar distribution to FS3, FS4, FS6, FS8, FS10, could not be tested in this assay since it did not give a sufficient signal in ELISA.

Antigen-modification studies Periodate oxidation. Mild periodate oxidation selectively denatures carbohydrate and so provides good preliminary evidence of whether MAbs are binding carbohydrate epitopes (Woodward et al. 1985). An E L I S A was used to quantify the extent to which periodate treatment of antigens reduced the ability of antibody to bind. The effects of periodate closely paralleled the k n o w n distributional patterns of antibody binding. Binding of MAbs

. . .









--33.3 6.7 --10.6

FS7 FS12

--12.1 -

-- 5.3 --12.5

FS3, FS4, FS6, FS8 and FS10 was significantly reduced if the antigen was pretreated with periodate (Fig. 2). The antigen recognised by M A b FS10 proved particularly sensitive, with binding reduced to less than 25% of the control following 1 h o f periodate treatment and abolished completely by 4 h periodate oxidation. Binding o f FS6 and FS8 was reduced to less than 20% by 4 h of periodate oxidation, and abolished by 16 h periodate oxidation. The antibody-binding capacity of the antigens recognised by FS3 and FS4 was also reduced significantly, though was not abolished, even by 72 h periodate oxidation. The M A b FS9 did not give a sufficient signal with E L I S A to determine reliably the effects of antigen modification o f antibody binding, but using dot-blots the antigen recognised by FS9 was shown to be even more sensitive to periodate oxidation than that of FS10 (Fig. 3 a). In contrast to these effects, binding of MAbs FS2, FS5, FS7 and FS12 was not reduced by pretreatment of the antigen with periodate for up to 72 h (see Fig. 2 for typical data). Binding of FS11 was also significantly and consistently, though only slightly, reduced following periodate oxidation of lysate. The effects o f periodate on FS1 binding to antigens could not be tested by E L I S A due to a low signal. However, a strong signal was obtained using dot-blot or indirect immunofluorescence, but there were no effects of periodate treatment on antibody binding (data not shown). Protease treatments. Modification of antigen by protease

to the extent that M A b binding is reduced or abolished can be taken as evidence that the antigen has a protein component. Three protease treatments were employed, viz. trypsin digestion of sperm lysate immobilised on E L I S A plates, pronase digestion o f immobilised lysate and pronase digestion of lysate prior to immobilisation.


J.L. Jones et al. : Fucus sperm surface antigens



._.--~w-----" #

~100" 75, ~


o 50,

o c

E E 5O


E E 25.



O0 0

Incubation (hours) Fig. 2. Effect of periodate treatment of antigen on binding of MAbs. ELISA plates were coated with Fucus sperm lysate which was then exposed to periodate or acetate buffer (controls) for the appropriate time at 4~ in the dark. Following ELISA, mean optical densities were calculated for the four replicate wells, compared with that for control wells by a t-test and expressed as a percentage of the control mean (relative immunoreactivity). Effects significant at P=0.01 (FS3, 4, 6, 8, 10), P=0.05 (FS11), nonsignificant (FS5, FS12). FS2 and FS7 gave similar curves to FS5 and FS12, respectively (data not shown). , , FS3; e, FS4; A, FS6; m, FS8; ~, FS10; A, FSll; o, FS5; w, FS12

Fig. 3a, b. Effects of periodate and trypsin treatments of antigen on binding of M A b FS9. Fucus sperm lysate, immobilised on nitrocellulose, was exposed to: a periodate (1-24 h at 4 ~ C in the dark) or b trypsin (1 h at 37 ~ C), and M A b binding was detected using H R P - R A M I G followed by hydrogen peroxide and 4-chloro-naphthol. FS2 and FS10 were included as positive controls and a M A b to rat bone cells as a negative control (C)







Trypsin Digestion (min) Fig. 4. Effect of trypsin treatment of antigen on binding of MAbs. Plates were coated with Fucus sperm lysate which was then exposed to trypsin or PBS (control) for the appropriate time at 37 ~ C. Results were calculated as described in legend to Fig. 2. Effects significant at P = 0 . 0 0 1 for FS2 (o), FS5 (o), FSI1 (A), FS7 (m), FS12 (D); non-significant for FS3 ( . ) and FS10 (~), which also represent FS4, FS6 and FS8 for which data are not shown

The trypsin treatment only affected significantly the binding of MAbs FS2, FS5, FS7, F S l l and FS12 which recognise antigens located preferentially on the anterior flagellum (Fig. 4). It did not affect M A b binding to antigens located preferentially on the cell body (FS3, 4, 6, 8, 10) (Fig. 4). Treatment of lysate with trypsin resulted in a large ( > 7 0 % ) reduction in binding of MAbs FS7 and FS12 with the maximal effect not apparent until 1 h trypsination. Treatment of the lysate with trypsin for 2 h reduced the binding of MAbs FS2 and FS5 by about 50%, though much of this effect was apparent after only 15 min treatment. Binding of M A b F S l l to its antigen is reduced by over 60% if the antigen is treated for 15 min. This effect is exhaustive in that incubation of antigen with trypsin for further periods is without further effect. In a dot-blot assay, pre-treatment of lysate with trypsin did not affect the binding of FS9 to its antigen, though binding of FS2 (used as a positive control) was abolished (Fig. 3 b). FS1 binding was also not affected by trypsin treatment of antigens in a dot-blot assay (data not shown). Pronase treatment o f immobilised lysate resulted in almost total abolition of binding of MAbs FS2, FS5, FS7, F S l l and FS12 (Fig. 5a). Interestingly, binding of MAbs FS4 and FS6 to immobilised antigens was also reduced significantly by pronase treatment though that of FS3, FS8 and FS10 was not affected (Fig. 5 a). Pronase treatment of the lysate prior to immobilisation produced a similar pattern to that observed with the trypsin treatment, with binding of MAbs FS2, FS5 and FS11 reduced by 40-50% and binding o f MAbs FS7 and FS12 affected to a greater extent (Fig. 5b). Binding of MAbs FS3, FS4, FS6, FS8 and FS10 was not affected by this treatment.


J.L. Jones et al. : Fucus sperm surface antigens a













The objectives of the current study were to obtain information on the molecular nature o f the cell surface components recognised b y a set of MAbs which bind to Fucus sperm. F r o m these characterisation studies the MAbs can be classified into groups identical to those derived from immuno-localisation studies (Jones et al. 1988, summarised in Table 1). The preliminary characterisations for each group o f MAbs are considered below. It appears that at least eight of the twelve MAbs bind carbohydrate epitopes and that a total of ten different epitopes are recognised.

50 o



FS11 and FS12. However, analysis of a pretreated lysate by SDS-PAGE, showed degradation o f proteins in the Mr range 100-160 k D a (data not shown). Since such proteolytic effects cannot be distinguished from a/~-elimination glycan-cleavage reaction, the above results must be treated with caution. Lower concentrations of N a O H were without effect on antibody binding.



C 2 5 7 12 11 3 4 6 8 10 MAb Number (FS)

Fig. 5a-c. Effects of pronase and Endo-F treatment of antigen

on binding of MAbs. Results were all calculated as described in the legend to Fig. 2. a Digestion of immobilised antigen with soluble pronase. Plates were coated with Fucus sperm lysate which was then exposed to pronase for the appropriate time at 37~ C. Effects significant at P=0.001 (FS2, 5, 7, 11, 12), P=0.01 (FS4), P=0.05 (FS6), and non-significant (FS3, 8, 10). b Digestion of soluble antigen with immobilised pronase. Sperm lysate was incubated for 6 h at 37~ C with pronase beads or PBS (control), centrifuged and the supernatant coated on to plates for ELISA. Effects significant at P=0.001 (FS7, 12), P=0.01 (FS2, 5, 11), non-significant (FS3, 4, 6, 8, 10). c Endo-F digestion of immunobilised antigen. Plates were coated with lysate which was then exposed to Endo-F for 24 h at 37~ C. Effects significant at P=0.001 (FS7, 12), non-significant (FS2, 3, 4, 5, 6, 8, 10, 11)

Boiling. This did not significantly reduce MAb binding to antigens (data not shown). Endo-F treatment. This selectively removes N-linked glycans and digestion o f immobilised lysate with this enzyme resulted in a decrease, by over 75%, in the a m o u n t of FS7 and FS12 binding to antigen (Fig. 5c). Binding of the other MAbs was unaffected by such treatment of the lysate (Fig. 5 c). Mild alkaline hydrolysis. This results in cleavage of glycans O-linked through the/3-hydroxy amino acids serine and threonine, but not of glycans O-linked through tyrosine or hydroxylysine, or through hydroxyproline, or N-linked through asparagine. Binding of MAbs FS3, FS4, FS6, FS8 and FS10 was not affected by such pretreatment of the lysate (data not shown) but there were large reductions in binding o f MAbs FS2, FS5, FS7,

MAbs FS3, FS4, FS6, FS8 and FSIO. The antigens recognised by these five MAbs appear, by indirect immunofluorescence, to be more densely distributed on the cell body than on the flagella (Jones etal. 1988; Table 1). Taken as a whole the evidence indicates that these MAbs are directed at glycan epitopes of a 205-kDa glycoprotein. This antigen can be detected on Western blots following SDS-PAGE, indicating that there is a protein component. Further, MAb binding is markedly reduced if the antigen is treated with periodate but not when the antigen is treated with trypsin or pronase beads, or if the antigen is extensively boiled, indicating that the actual epitope recognised is carbohydrate. The effect of soluble-pronase treatment of immobilised lysate on binding of FS4 and FS6 provides further evidence that the antigen has a protein component. The lack o f effect of insoluble-pronase treatment on the antigens of these MAbs indicates that the effect o f soluble pronase results from detachment of glycan from the plate following digestion of the protein backbone, rather than a digestion of the epitope itself. The extent of the glycosylation of the antigen recognised by this set o f MAbs would appear to be variable, giving rise to the broad, diffuse band in the Western blot. However, the nature o f the glycanpeptide linkage remains uncertain in that Endo-F digestion and/%elimination of antigens had no effect on antibody binding. In addition, gum arabic from Acacia senegal had no effects in competition-binding assays (data not shown), indicating that the 205-kDa antigen is not an arabinogalactan protein. The combined evidence of immunofluorescence, Western blots and the additivity assays can be taken to indicate that the MAbs FS3, FS4, FS6, FS8, FS10 bind to the same antigen. That this set of five MAbs were initially chosen from a large number which showed similar binding patterns using indirect immunofluorescence, indicates that the 205-kDa glycoprotein is an im-

70 munodominant antigen on the sperm cell surface. The results of the additivity assay might also be taken to indicate that these five MAbs each recognise the same epitope. However, this seems unlikely. For example, FS10 is evidently not duplicated since it is the only MAb that binds sperm in a strong species-preferential manner. Furthermore, FS4 binds to eggs of F. serratus and to sperm of Ascophyllum nodosum, whereas the other MAbs (FS3, FS6, FS8, FS10) appear not to (Jones et al. 1988). It also seems unlikely that FS3, FS6 and FS8 recognise exactly the same epitope because of differences in epitope sensitivity to periodate and the fact that FS6 binding is reduced following soluble-pronase treatment of antigens whereas FS3 and FS8 binding is not affected. The competition exhibited by the MAbs in the additivity assay is evidently not an artefact of the system since none of the MAbs compete with FS11, a MAb known to bind to a distinct epitope and which was thus used to validate the system. The most likely explanation of the results is that these five MAbs each recognise a different epitope, but that these epitopes are neighbouring or overlapping. Consideration of the relative sizes of an immunoglobulin (IgG= Mr 150 kDa and IgM =Mr 900 kDa) and an epitope of perhaps two to six monosaccharide units (approx. 400-1 200 Da) emphasises the likelihood of steric hinderance occurring between two MAbs attempting to bind neighbouring epitopes. In binding to sperm cells the MAbs FS3, FS4, FS6, FS8 and FS10 exhibit different taxonomic specificities (Jones et al. 1988) which probably reflect differences in the carbohydrate side chains of the 205-kDa glycoprotein as described above. This situation may be analogous to that in Chlamydomonas eugametos gametes, in which strain-specific epitopes are correlated with the presence of certain O-methylated sugars in the isoagglutinin glycoproteins (Homan et al. 1987; Schuring et al. 1987). MAbs FS9 and FS1. FS9 labels the entire sperm body, including both flagella, and this antibody also cross reacts with eggs, so that it has a similar binding distribution to FS4. However it was not possible to detect any antibody binding in Western blots, and the possibility that FS4 and FS9 bind the same epitope can be excluded on the basis that the binding of the two MAbs is affected to differing extents by modification of the antigen with periodate. Also, FS9 gives a much weaker signal in ELISA than does FS4, and it is unlikely that this is due entirely to lower affinity since it gives a high signal in other assays such as immunofluorescence and dot-blot. Unfortunately it was not possible to characterise the antigen recognised by MAb FSI, which appears to bind primarily to the sperm cell body, because the MAb failed to Western-blot and gave a poor signal in ELISA. Using immunofluorescence and dot-blot, it was still not possible to demonstrate sensitivity of this antigen to periodate or protease (data not shown) so the true nature of this antigen remains unknown. It is possible that the MAb recognises an epitope of a glycolipid. MAbs FS7 and FS12. These two MAbs, which recognise an epitope present on the mastigonemes, the cell body

J.L. Jones et al. : Fucus sperm surfaceantigens and posterior flagellum (Jones et al. 1988), show identical labelling patterns on Western blots, exhibit similar characteristics in all the antigen-modification assays and compete under conditions of limited antigen availability. This implies that they are directed at the same epitope, a conclusion further supported by the fact that neither MAb cross reacts with Fucus eggs nor binds to sperm in a species-preferential manner (Jones et al. 1988). The antigens to which these two MAbs are directed fall into the Mr range 40-250 kDa. Binding of these MAbs is markedly affected if the antigen is treated with protease or Endo-F, indicating that the antigens have both a protein and an N-linked carbohydrate component. On the basis that the MAbs label multiple bands in Western blots and that the antigen is generally insensitive to extensive boiling it is likely that the epitope recognised by the antibodies is an N-linked carbohydrate moiety common to a number of glycoproteins. The effects of mild alkaline hydrolysis can not be interpreted as indicating the presence of O-linked glycan since the treatment also causes proteolysis. Binding of FS7 and FSI2 is resistant to periodate oxidation treatment of antigens, but this could result from the protection of particular hydroxyl groups in the sugars of the glycan chains (Fry 1988). MAbs FS2, FS5 and F S l l . These three MAbs recognise epitopes found on the anterior flagellum and they bind to proteins in Western blots with Mrs in the range 90250 kDa. FS2 and FS5, which label mastigonemes (anterior flagellum) and patches on the cell body (Jones et al. 1988), show identical labelling patterns on Western blots, exhibit similar characteristics in all the antigen modification assays and compete under conditions of limited antigen availability. This indicates that they are directed at the same epitope, and this is supported by the fact that both MAbs cross react with Fucus eggs and bind to sperm in a genus-preferential manner (Jones et al. 1988). FS11 binds an epitope present only on the plasma membrane of the anterior flagellum (Jones et al. 1988) and has a different labelling pattern from FS2 and FS5 in Western blots. The antigens recognised by FS2, FS5 and F S l l appear to have protein components since binding of MAbs is markedly affected by treatment of the antigens with trypsin or pronase. However, binding of these MAbs is either unaffected or only slightly affected by Endo-F or periodate-oxidation treatment of antigens and the effects of mild alkaline hydrolysis can not be interpreted as being due to proteolysis. Overall it is not possible at this stage to say whether these antigens are glycosylated. That many of the MAbs appear to recognise carbohydrate is perhaps not surprising, given that this is typical of previous studies in which MAbs have been produced to plant cell surface antigens (Smith et al. 1984; Bradley et al. 1988; Key and Weiler 1988). Our studies show that the surface of Fucus sperm exhibits a number of proteins/glycoproteins, including an immunodominant 205-kDa antigen. The evidence indicates that these antigens are held in distinct spatial arrays and has implications both in differentiation and in cell surface mainte-

J.L. Jones et al. : Fucus sperm surface antigens nance. W h e t h e r this holds a n y parallel with the c o n c e p t o f d o m a i n s in a n i m a l cell systems, a n d p a r t i c u l a r l y w h e t h e r there is a n a n a l o g y with respect to f u n c t i o n , r e m a i n s to be d e t e r m i n e d . We are grateful to AFRC for financial support under the 'cell signalling' initiative.

References Bradley, D.J., Wood, E.A., Larkins, A.P., Galfre, G., Butcher, G.W., Brewin, N.J. (1988) Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum. Planta 173, 149-160 Friguet, B., Djavadi-Ohaniance, L., Pages, J., Bussard, A., Goldberg, M. (1983) A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognise the same antigenic site. Application to hybridomas specific for the fl2-subunit of Eseherichia coli tryptophan synthase. J. Immunol. Methods 60, 351 358 Fry, S. C. (1988) The growing plant cell wall: chemical and metabolic analysis, pp. 135-137. Longman, London Homan, W.L., van Kalshoven, H., Kolk, A.H.J., Musgrave, A., Schuring, F., van den Ende, H. (1987) Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognise strain-specific O-methyl sugars. Planta 170, 328-335 Horst-Walker, B., Koepsell, H. (1987) Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding. Anal. Biochem. 164, 12-22 Jones, J.L., Callow, J.A., Green, J.R. (1988) Monoclonal antibodies to sperm surface antigens of the brown alga Fucus serra-

71 tus exhibit region-, gamete-, species- and genus-preferential binding. Planta 176, 298-306 Key, G., Weiler, E.W. (1988) Monoclonal antibodies identify common and differentiation specific antigens on the plasma membrane of guard cells of Viciafaba L. Planta 176, 472481 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Primakoff, P., Hyatt, H., Tredick-Kline, J. (1987) Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion. J. Cell Biol. 104, 141-149 Saling, P.M., Lakoski, K.A. (1985) Mouse sperm antigens that participate in fertilisation. II. Inhibition of sperm penetration through the zona pellucida using monoclonal antibodies. Biol. Reprod. 33, 527-536 Saling, P.M., Irons, G., Waibel, R. (1985) Mouse sperm antigens that participate in fertilisation. I. Inhibition of sperm fusion with the egg plasma membrane using monoclonal antibodies. Biol. Reprod. 33, 515-526 Schuring, F., Smeenk, J.W., Homan, W.L., Musgrave, A., van den Ende, H. (1987) Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos. Planta 170, 322-327 Smith, E., Roberts, K., Hutchings, A., Galfre, G. (1984) Monoclonal antibodies to the major structural glycoprotein of the Chlamydomonas cell wall. Planta 161, 330-338 Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354 Trimmer, J.S., Trowbridge, I.S., Vacquier, V.D. (1985) Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca z§ and H § fluxes of sea urchin sperm. Cell 40, 697-703 Woodward, M.P., Young, W.W., Bloodgood, R.A. (1985) Detection of monoclonal antibody specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78, 143-153

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12.

Sperm of the brown alga Fucus serratus are highly differentiated, biflagellate, naked cells. Immunolocalisation studies, employing monoclonal antibodi...
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