developmental changes in the structural organization of the lectin discoidin i detected by limited...

Vol. 152, No. 3, 1988

May 16, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1332-1338

DEVELOPMENTAL CHANGES IN THE STRUCTURAL ORGANIZATION OF THE LECTIN DISCOIDIN I

DETECTED BY LIMITED PROTEOLYSIS

Alfonso Valencia, Angel Pestafia and Amparo Cane

Institute de Investigaciones Biom~dicas, CSIC y Departamento de Bioquimica de la Facultad de

Medicina. U,A.M. Arzobispo Morcillo, 4. 28029 Madrid. Spain

Received March 30, 1988

Digestion of discoidin I with several proteolyiic enzymes reveals the existence of structural domains in this lectin. Significative differences have been detected in the pattern of fragments generated by V8 protease on discoidin I of various developmental situations. The changes observed can be related to the presence of various types of tetrameric structures in discoidin I. Together with the presence of different types of isoforms in vegetative vs. differentiated cells, the results presented here suggest the involvement of different structural organizations in discoidin I which can be related to the biological functions of this lectin. ® 1988 Aoademic Press, Inc.

The N-actylgalactosamine binding lectin, discoidin I, has been implicated in

the aggregation of the slime mold Dictyostelium discoideum at the level of intercellular

adhesion (1) and, in similarity with fibronectin, in cell-substratum attachment and ordered

cell migration (2). Discoidin I is constituted by a family of three different isoforms (M.W.

30 Kd) with isoelectric points of 6.9, 6.8-6.7 and 6.2 (3) encoded by a family of four or

five closely related genes (4,5). The expression of discoidin I is developmentally regulated at

the level of transcription (6,7), and, at least three of the members of the gene family are

coordinately expressed in the three isoforms during development (3,5). However,

vegetative cells growing in axenic medium do not express one of the discoidin I genes and,

consequently, they do not accumulate the 6.8-6.7 isoform (3).

The active form of discoidin I has been described as a tetramer of about 30 Kd

monomers several years ago (8), however very little is still known about the relationship

between structural organization and the different functions adscribed to discoidin I of

differentiated cells as well as with the apparent functionless of this protein in vegetative

cells.

Limited proteolysis is an approach commonly used to study the organization of

proteins in structural domains (9), as well as to detect even subtile differences between

proteins of different sources or tissues, as in fibronectin(10,11). We have applied this

methodology to discoidin I purified from vegetative cells and from different developmental

states in order to investigate the possible domain organization of this multimeric lectin. The

results presented in this communication show for the first time the existence of variability

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 1332

Vol. 152, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the structural organization of discoidin I from various developmental situations. This new

data can help in our present understanding of the functional role(s) of this protein.

MATERIALS AND METHODS

Strain. arowth and develoomental conditions. Dictyostelium discoideum, axenic strain AX-2, was grown in HL-5 medium as

previuosly described (1). Development either in suspension or on solid support (on Millipore filters) as well as discoidin I purification from different developmental situations was carried out as previously described (1)

Proteolvtic diaestion of discoidin I. Limited proteolytic digestions of the indicated amount of purified discoidin I

samples (1 mg/ml) were carried out in 20 mM Tris-HCI pH 7.0 with 10% (w/w) concentration of the indicated proteases. Reactions were allowed to proceed at 37°-C and were stopped at different times by boiling for 2 rain in sodium-dodecyl-sulphate (SDS)-electrophoresis sample buffer(12).

Analysis of diaestion, determination of the size of the fraaments and Quantitation.

The different fragments generated by the action of the proteases were analysed by SDS-polyacrylamide gel electrophoresis on 15% or 10-20% gradient slab gels as described by Laemmli (12). Gels were stained in 0.02% Coomassie blue in Methanol:Acetic acid (20%:10%). The sizes of the monomer and of different fragments were calculated from the migrations obtained in the gel as compared with those of known molecular weigth markers, using the plot: log of migrated distance in cm, vs. square root of molecular weight. The variation in the apparent M.W. obtained in different gels with this method was 5%.The values presented are the mean of at least three individual determinations.

Quantitation of the different fragments was carried out by weighting the areas obtained in densitometric scans (Elisa Quick Scan densitometer) of the stained gels.The results presented are the mean of two independent weightings from duplicated scans.

Other analvtical orocedures. Protein concentration of discoidin I samples was determined by the Bradford

procedure (13) using bovine serum albumin as standard. Proteolytic enzymes, Trypsin (E.C.3.4.21.4), Elastase (E.C.3.4.21.36),

Staphilococcus aureus V8 protease (protease type XVII from S.aureus, strain 8) and low molecular weight markers were supplied by Sigma Chemical Co. Electrophoresis reagents were from BioRad. The rest of reagents were of the highest purity available.

RESULTS

Diqestion of discoidin I with different proteases.

Discoidin I purified from cells developed during 9h in shaken suspension, T9 s,

was subjected to limited digestion with several proteolytic enzymes. As can be seen in Fig.l, a

similar pattern of fragments were released from discoidin I under, the action of three

different proteases: a group of fragments slightly smaller than discoidin I monomer (from

29.0 to 27.0 Kd) and another group of small fragments (from 17 to 12 Kd). Two of these

smaller fragments (17 and 15.0 Kd)are released by the three proteases (see arrows in

Fig.l) and together they account for the whole size of the monomer (32 Kd) . These data,

together with the inspection of the known aminoacid sequence of discoidin I (5), suggest the

origin of the small fragments from a single cut in the center of the monomer.The larger

fragments detected (29.0 to 27.0 Kd) should came from trimming from the ends of the

monomers. Secondary digestions of isolated fragments from trypsin and S.aureus V8 digest

1333


IVlW (Kd)

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Fig.l. Digestion of discoidin I with several oroteases . 25 ~tg of discoidin I from T9 s cells were incubated at 37-°C with 10% (w/w) of the follgwing proteases :1, elastase (2h), 4, trypsin (2h) and 7, S.aureus V8 protease (8h). The digestion products were analysed on SDS 10-20% polyacrylamide slab gels. Lane 2 and 3 show elastase control at 1% and 10% respectively, regarding to discoidin I. Lanes 5 and 6, trypsin control, 10% and 100%, repectively. Lane 8, S.aureus V8 protease control, 10%. Lane 9 discoidin I control (15.ttg) incubated for 2h at 37-°C without proteases. M, low molecular weigth markers, with sizes indicated on the left of the figure in Kilodaltons (Kd). Migration of discoidin I (DI) as well as that of frgaments of 17 and 15 Kd are indicated by arrows.

Fig.2. Time course digestion of discoidin I with trvosin. 40~g of discoidin I from T9s ceils were incubated with 10% (w/w) of trypsin at 37-°C during 5min (lane 3), 15min (lane 4), 30min (lane 5), 60rain (lane 6) and 120min (lane 7). Lanes 1 and 2 shows trypsin controls at 100% and 10% w/w regarding to discoidin I. M, stands for molecular weight markers. A. Coomassie blue stained gel. Arrowheads indicate the migration of trypsin peptides. B. Densitometric scanning of the indicated lanes of the gel.

with cyanogen bromide (14) (based in the presence of only one Met at position 72 in

discoidin I sequence) show that the group of small fragments arises from a single cut in the

middle of discoidin I monomers, giving rise to the 15 and 13 Kd fragments from the

N-terminal part and to the 17 Kd fragment from the C-terminal part of the molecule,

whereas those close to 28 Kd arise from a single cut at the C-terminal part of discoidin I

molecules (not shown).

Time-course of trypsin diqestion.

A time-course digestion of discoidin I from T9 s cells with trypsin (Fig, 2)

shows that the group of small fragments are clearly detected after 15 min and remain

without any significative change up to 60 min digestion, whereas the total amount of the

larger ones shows a progressive decrease as digestion proceeds. In Fig.2B the corresponding

scannings of the gel of Fig.2A are represented, showing that there is a relative progressive

increase in the amount of the 27Kd fragment at the expense of the larger ones of 29 and 28 Kd

as digestion proceeds; in contrast, no changes are detected either in the total or relative

amounts of the small group of fragments up to 60 min digestion (diagrams 3 to 6, Fig.2B)

which suggests a steady-state situation.

At longer digestion times,120 min (lane 7 in Fig.2A), all the fragments show

a marked reduction in amount, such decrease should reflect the subsequent proteoiytic

degradation of the fragments However, no other intermediate fragments are generated

indicating the existence of resistant cores inside the monomers; this fact probably reflects

1334

A

Vol. 152, No. 3, 1988

I M 2 M

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4 5 M

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Fia.3. Diaestion of discoidin I from different develoomental staqes with Staphilo0occus aureus V8 orotease. 30 I.tg of discoidin I samples were digested with 10% (w/w) of S.aureus V8 protease at 37gCduring 6h. A. Electrophoretic analysis of the fragments generated on SDS-15% polyacrylamide slab gel, the different lanes correspond to digested discoidin I from the indicated developmental times on the top of each one. M, stands for molecular weigth markers. The migration of discoidin I (DI), discoidin II (DII) and that of characteristic fragments are indicated on each side of the gel. B. Quantitation of the results shwon on panel A. The amount of each fragment relative to the total estimated protein on the scan of each lane of the gel is represented. Only those fragments representing more than 1% of the total estimated protein on each scan are presented. C. Comparative digestion of purified discoidin I and II with V8 protease during 6 h. Migration of both proteins and of fragments generated are indicated.

the high degree of compactation of discoidin I molecules, and supports the existence of

structural domains, corresponding to the fragments detected, in the native molecules, as in

another proteins (15).

On the other hand, the origin of the two group of fragments from discoidin I has

been unambigously determined by Western blotting and immunoprecipitations of digest with

anti-discoidin I antibodies (not shown).

Digestion patterns of discoidin I purified from different developmental states

qenerated bv Staphilococcus aureus V8 Drotease.

We next investigated the pattern of fragments generated by limited

proteolysis on discoidin I purified from cells developed on filters during 3 (T3), 6 (T6)and

9h(T9)or in suspension during 9 h (T9s), as well as from vegetative cells while growing in

exponential phase (TO). Fig.3A shows the patterns of fragments obtained from the different

1335


samples after digestion with V8 protease during 6h. As can be observed noticeable differences

were detected with the various discoidin I preparations:

1. Discoidin I from TO cells only generates fragments of high M.W. (28.6,a, and 27 Kd,b)

(lane1 ,Fig.3A).

2. Discoidin I from different developmental times generates both high and low M.W.

fragments, however a different pattern of fragments is obtained from discoidin I of cells

developed in suspension and on solid support. Such differences refer both to the sizes and

abundance of the fragments generated (compare lane 2 with 3 to 6, Fig.3A).

3. Discoidin I from cells developed for different times on filters generates the same pattern of

fragments: the most abundant being one of 24 Kd (d) and another one of 17 Kd (k) (lane 6,

FIg.3A). Both fragments are detected at the three developmental times tested, but they show a

marked tendency to decrease in abundance as development proceeds.

4. Discoidin I from T9 s cells in contrast generates a fragment of 28 Kd (b) very abundant, as

well as a series of smaller fragments ranging from about 21.5 Kd (e) to 14 Kd (n); the most

abundant of this series being those of 19Kd(h), 15.4Kd(m) and 14Kd(n). Of them only that

of 15.4 Kd (m) appears also on discoidin I from cells developed on filters.

Fig.3B shows a quantitation of the results of Fig.3A. From this representation

the decrease in fragments abundance as development proceeds, as well as the quantitative and

qualitative differences detected in the patterns of discoidin I from cells in different

developmental situations can be clearly observed (compare panel d with the others, Fig.3B).

It has to be pointed out that the content of discoidin II in the different

preparations varies from one another (12% for TO, 5.4% for T3, 7.5% for T6, 7.2% for T9

and 3.5% for T9 s , as percentage of the total protein content of the preparations). However

the content of discoidin II does not influence the pattern of fragments generated by V8

protease : a) Purified discoidin II remain mostly undigested by this protease after 6h

digestion, and none specific fragment with apparent mobility of those shown in Fig 3A can be

detected in our analytical system (Fig.3 C). b) The absolute amount of fragments d (24Kd)

and k (17Kd) detected on preparations from cells developed in filters, after 6h digestion, far

exceeds the initial content of discoidin II in such preparations; this fact is specially relevant

in T3 preparations (lane 5, Fig.5A and panel T3 in Fig.3B) where the initial content of

discoidin II is 5.4 % and the amount of fragment d generated amounts to a 32% of the total

digested protein.

DISCUSSION

The structural organization of discoidin I has been studied through limited

proteolysis. Discoidin I appears to be organized in structural domains which can be released

as protected fragments after limited proteolysis with several proteases. Most of the

fragments generated persist even after long digestion times with high concentrations of the

proteases suggesting a high degree of compactation of discoidin I molecules.

The kinetic data, shown in Fig.2, clearly support the origin of the two group of

fragments from independent populations of discoidin I molecules. In this context, it is

1336


specially relevant to remark the absence of the group of small fragments in discoidin I from

vegetative cells. This fact appears to be related to the lack of expression of 6.7-6.8 isofrom

in this cell population (3), where the existing two isoforms should be organized in different

types of tetramers than those present in differentiated cells (cross-linking studies support

the existence of tetramers the protein of vegetative cells, not shown). The specific structural

organization of discoidin I in vegetative cells may be related with the apparent funtionless of

discoidin I in this cell-cycle stage of D.discoideum.

The differences detected in the pattern of fragments generated from discoidin I

of various developmental situations suggest the possible existence of various types of

multimeric organizations in discoidin I molecules. During the first hours of development, the

relative content of the three isoforms should experiment changes concomitant with the

expression of the 6.8-6.7 isoform , which should be reflected in the appearance of new kind

of tetramers not present in vegetative cells. This fact could partly explain, by itself, the

differential susceptibility to V8 digestion and the accumulation of different fragments

observed from vegetative cells to increasing developmental times. However,the differences

observed between cells developed on filters and in suspension, after 9h starvation, can not be

explained by variations in the three isoforms, as they are coordinately expressed during

development (3). Such differences must be related to differential accesibility of specific

regions in the various isoforms, probably organized in different kind of tetramers in both

developmental situations.

Another possibility would be that the overall multimeric structure be

differentially affected in both situations by the existence of postranscriptional modifications .

Even subtile modifications could result in profound effects in the multimeric structure if

they affect to interacting regions of the monomers,as have been reported in other proteins,

such as tubulin, where the phosphorylation of the J~-subunit results in a completely different

pattern of tryptic fragments(16).

The differences in the structural organization of discoidin I from various cell

populations, reported here for the first time, suggest the association of specific tetrameric

organizations with the different functions adscribed to this lectin. For instance, during

development in suspension the main function of discoidin I should be related with

intercellular adhesion, in contrast to filter developed cells where cell-substratum

attachment can play a major role in the morphogenetic process (2). The differential

susceptibility to proteolysis detected in discoidin I can reflect the prevelance of specific

structural/functional organizations in the various cell populations analyzed.

ACKNOWLEDGEMENTS

We deeply appreciate the technical assistance of A. Montes in the maintenance of Dictyostelium cultures and in the purification of discoidin. The assistance of A. Fernandez and A. Diaz in the preparation of graphics and photography of the manuscript is also acknowledged. With support of research grants from the Comisi6n Asesora Investigaci6n Cientifica y T~cnica (CAICYT) and Fondo Investigaciones Sanitarias (FIS). A. Valencia is a predoctoral fellow from the Consejo Superior de Investigaciones Cientificas (CSIC).

1337


REFERENCES

(1) Cano, A. & PestaSa, A. (1984)J.CelI.Biochem. 25, 31-43. (2) Springer, W.R.; Cooper, D.N.W. & Barondes, S.H. (1984)Cell, 39, 557-564. (3) Devine, J.M., Tsang, A.S. & Williams, J.G. (1982) Cell, 28, 793-800. (4) Rowekamp, W. Poole, S. & Firtel, R.A. (1980) Cell, 20, 495-505. (5). Poole, S., Firtel, R.A., Lamar, E. & Rowekamp, W. (1981) J.MoI.Biol., 153, 273-289. (6) Ma, G.C.L. & Firtel, R.A. (1978) J.Biol.Chem., 253, 3924-3932. (7) Williams, J.G., Lloyd, M.M. & Devine, J.M. (1979) Cell, 17, 903-913. (8) Simpsom, D.L.; Rosen, S.D. & Barondes, S.H. (1974) Biochemistry, 13, 3487-3493. (9) Rotwell, W.S, Grasser, A.W & Murphy, B.B. (1986) Annual N. Y. Acad. Sciences. 466,

103-110 (10) Sekiguchi, K., Siri, A., Zardi, L. & Hakamori, S. (1985)J.Biol.Chem., 260,

5105-5114. (11) Zardi, L.; Carnemolla, B.; Siri, A.; Petersen, T.E.; Paolella, G.; Sebastio, G. and

Barrele, F.E. (1987) EMBO J., 6, 2337-2342. (12) Laemmli, U.K. (1970) Nature, 277, 680-685. (13) Bradford , M.M. (1976) Anal. Biochem., 72, 248-254. (14) Nikoden, V. and Fresco, J.R. (1979) Anal. Biochem., 97,382-386. (15) Wagner, D.D. and Hynes, R.O. (1979) J.Biol. Chem., 254, 6746-6754. (16) Wandosel, F, Serrano, L, Hernandez, M.A & Avila, J. (1986). J.Biol. Chem.,261,

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developmental changes in the structural organization of the lectin discoidin i detected by limited...

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