Aggregation of sponge cells: a novel mechanism of controlled intercerllular adhesion

Aggregation of sponge cells: a novel mechanism of controlled intercellular adhesmn Werner E. G. Miiller and Peter Vaitht Institut j~r Physiologische Chemie, Ahteilun,q An,qewandte Molekularhioloqie, Universit~it, Dueshergweq, 6500 Mainz, W. Germany ? Medizinische UniversitYttsklinik, Abteihmq Experimentelle Medizin, Kerpener Strasse 15, 5000 Kbln, W. Germany

(Received 8 June 1978; revised 14 September 1979) From the marine sponge G e o d i a c y d o n i u m a series of macromolecules have been isolated and characterized which are involved in the control o]ag.qre.qation and separation o[sponye cells: these include a q.qregation]~wtor, a.qgregation receptor, anti-aggregation receptor, [3-.qlucuronidase, [~-glucuronosyltran~[erase, [Jgalactosyltrans~erase, [J-yalactosidase and a lectin. These components might be linked in the ]ollowing sequence: (a) activation of the a,q,qregation receptor by its enzymic glucuronylation: (b) adhesive recognition qf the cells, mediated by the aggregation Jactor and the glucuronylated ag(tregation receptor; it) inactivation ~f the a q,qregation receptor by its de qlucuronylation with the membrane-associated [~-glucuronidase: (d) cell separation due either to the loss of the recognition site (ylucuronic acid) of the a qgre(lation receptorJbr the a q,qregation,]actor or to an inactivation of the aqgregation .]actor by the anti-a~Lqregation receptor. The activity ~?] the antia qgregation receptor is probably controlled hy the G e o d i a lectin.

Introduction The evolution of mesozoan-, parazoan- and eumetazoan organisms required the development of specific mechanisms to coordinate cell division, cell movement and cell-cell interactions, resulting in differential gene expression. These mechanisms are interrelated, and they are controlled by an assembly of interacting macromolecules which, first, are located on the cell surface, secondly, span the cell membrane and, thirdly, are associated with submembranous structures. This transmembranous control system is not static, but its components are embedded into a cell membrane, which is a fluid structure ~. Failure of this control system to function correctly with respect to time and cell type may result both in a maldifferentiation of the respective cell during embryonic and adult development and in an altered intracellular activity, e.g. in a phase of untimely gene expression. Studies to elucidate the biochemical mechanisms of cell~cell interactions are restricted to model systems which are based on reaggregation of dissociated cells in vitro. We have chosen the marine cosmopolitan sponge species Geodia cydonium Jam. The sponge cells were routinely dissociated chemically in calcium- and magnesium-free artificial seawater (CMF) containing trypsin 2. After being transferred into calcium- and magnesium-containing artificial seawater (ASW) the cells start to reaggregate. The aggregation process can be subdivided into three phases. First, the formation of Abbreviations: AF, aggregation factor; CPP, circular proteid particle; AR, aggregation receptor; aAR, anti-aggregation receptor; CMF, calcium- and magnesium-free artificial seawater; ASW, calcium- and magnesium-containing artificial seawater * This paper is dedicated to Professor Gerd Uhlenbruck on the occasion of his 50th birthday.

0141 8130/80/020093 04502.00 © 1980 IPC Business Press

primary aggregates; secondly, the formation of secondary aggregates; thirdly, the reconstitution of a functional sponge 3. This paper describes the subcellular events during the aggregation of Geodia cells. The macromolecules known to be involved in aggregation are listed and the functions of these macromolecules during the process of cell recognition are summarized.

Cell-aggregating factors The extracellular macromolecules which are of interest in studies of cell-cell interactions in the Geodia system are localized on the circular proteid particles.

Circular proteid particle: ylycosyltransfeases

aggregation

Jiwtor

and

The circular proteid particles (CPPt, have been purified to homogeneity z'4. They are characterized by a sedimentation coefficient of 90 S (mol. wt. 1.3 × 108)5'° and can be visualized by electron microscopy 6'7. The entire particles appear as spheroidal structures (major axis: 1125,8,; minor axis: 248 £); after treatment with detergents, core structures consisting of a central circle (contour length 3530 £) and 25 radially arranged filaments (length 610 ¢/) are observed. The C P P are composed of protein (74~,), lipid 0 (/o), carbohydrate (10~o) and inorganic material 6. Up to three known functional subunits of the C P P have been identified: aggregation factor and two glycosyltransferases 4'8.

Aggregation factor This has been obtained in soluble form after treatment of C P P with non-ionic detergents 4. The AF consists of

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Aggregation qJsponge cells: 14"erner E. G. Miiller and Peter Vaith 911~,, protein (with lysine and arginine as terminal amino acids) and 61'0 carbohydrates. Its sedimentation coefficient was determined to be 2.7 S, corresponding to a molecular weight of 23 000.

Glueuronosyltran.~Jerase This glycosyltransferase s uses deglucuronylated aggregation receptor (AR) from the same species as acceptor with an affinity [Kin) of 216 riM. The enzyme is further characterized by a K,, value for UDP-glucuronic acid of 1.I mM. fi-t~-Galactosyltran,~lorase The enzyme s was found to use both degalactosylated AR and degalactosylated anti-aggregation receptor (aAR) as acceptors. The K,, values for the two acceptors differ greatly: AR was determined to have a K,, of 660 nM, while a value of only 0.28 nM was calculated in the case of the aAR. This finding indicates that the aAR is the preferred acceptor, probably also under physiological conditions on the cell surface. The K,,, values for the sugar substrate (UDP-galactose) were found to be between 0.1 and 0.4 mM for both substrates. A series of cell surface molecules were isolated from Geodia cydonium, some of which also have enzymic activity. Aggregation receptor The aggregation receptor (AR) has been isolated and purified 9. It sediments at about 2.6 S, corresponding to a molecular weight between 15 300 and 18 000. The buoyant density was determined to be 1.51 g cm 3. The chemical properties of the AR are well documented 1°. A R contains 6.10o protein and 53.5'!,o carbohydrates. Three major sugars (D-galactose, D-glucose and D-glucuronic acid) account for ~ 85°o of the total carbohydrate. From biological, as well as enzymic, experiments strong evidence exists that D-glucuronic acid is the terminal sugar moiety which reacts with the AF. Anti-aggregation receptor The anti-aggregation receptor (aAR) has recently been isolated and purified from cell membranes of Geodia cydonium~: it reversibly inactivates the AF. This molecule was characterized as a glycoprotein (54'}; neutral carbohydrate, 3.2'!; hexuronic acid, 7.9,°/; lipid and 17.8",; protein) with a molecular weight ~ 180 000 and a buoyant density of 1.43 g cm 3 One biologically active site of the aAR was determined to be D-galactose. The aAR interferes with the A F A R complex. Leetin The lectin was isolated to homogeneity from a crude AF preparation {extracellular fluid) by affinity chromatography lz. It is a glycoprotein, heat labile, Ca 2+independent and resistant to EDTA. The molecular weight of the lectin was determined to be 72 000 and it consists of six subunits (mol. wt. 12000). The lectin is specific for fi-linked D-galactose residues and for Nacetyl-D-galactosamine. We have found that the lectin precipitates with the aAR ~3

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fi-D-Glucuronidase The cell membrane associated fi-D-glucuronidase ~'~has a broad pH optimum between 6.5 and 4.0. Both the divalent cations Mg 2 + and Ca 2 +, as well as EDTA, have no effect on the enzyme reaction. For full enzyme activity 200 mM NaC1 must be added to the reaction mixture. The temperature optimum was determined to be ~ 2 0 C , and the temperature coefficient, Q~ 0, 2.6. The apparent molecular weight was estimated to be ~ 80000. fi-D-Galact osidase The fl-D-galactosidase is cell membrane associated 1'~, has a molecular weight ~ 25 000 and a pH optimum ~ 5.0. The enzyme activity does not require the presence of Mg 2+, Ca 2. and EDTA but is dependent on a salt concentration of 200 mM. The temperature optimum is ~ 2 2 C, and the Q~0 value was determined to be 2.9.

Molecular events during aggregation The aim of this section is to present a unifying concept of the combined functions of the known non-enzymic and enzymic macromolecules which are probably directly involved in cell recognition in the Geodia system.

Cell aggregation and cell separation (via activation or inactivation o.[ AR) Stimulated by the studies of Burger 15 we have also succeeded in isolating an AR. This molecule is the recognition molecule for AF 9 but it is not involved in the process of primary aggregation 9. The requirement of the AR for the AF-mediated cell reaggregation is well established9: e.g. AR-depleted Geodia single cells show a lagphase > 6 0 rain 9 until they start to form secondary aggregates. This lag-phase can be shortened considerably by the addition of isolated AR to the single cell suspension, then the time to the onset of secondary aggregation is only 30 min. Incubation of the cells with heterologous flglucuronidase (from E. coli) reduces the aggregation potency of the cells considerably I°. In a recent study 14 it was established that the cell membrane associated homologous fi-glucuronidase from Geodia also exerts the same effect. The direct evidence that the cell membrane associated fl-glucuronidase removes terminal D-glucuronic acid moieties from AR was performed with an AR preparation, labelled in vivo with D-[14C]glucuronic acid ~4. After incubation of the labelled AR with membrane-associated fl-glucuronidase, only 65?o of the original radioactivity could be recovered. The deglucuronylated AR can, under certain physiological conditions, be enzymatically reglucuronylated by the CPP-linked glucuronyltransferase 14. From these experimental findings the following working hypothesis to explain cell aggregation and cell separation on the basis of an interaction between the glycosidase (fl-glucuronidase) and the glycosyltransferase (glucuronosyltransferasel can be formulated (Figure 1). (a) Activation of the AR by its enzymic glucuronylation. (b) Adhesive recognition of two cells, mediated by the AR and the glucuronylated AR. (c) Inactivation of the AR by its deglucuronylation with membrane-associated fi-glucuronidase. (dl Cell separation due to the loss of the recognition site (glucuronic acid) of AR for AF.

A.qflreqation o/sponge eel~s: Werner E. G. Midler am/Peter Vaith

~

(activotion of AR)

~ ~

GluA - transferose

IV/\ v

The binding sites involved in the formation of the A R AF complex are D-glucuronic acid, linked to the AR 1°, and L-lysine, the terminal amino acid of the AF +'~°.

UDPGluA

UDP

UDP-O

Cell separation

Adhesive

(inactivation of AR)

recognition

F,

Figure I Proposed molecular mechanisms for cell aggregation and cell separation via activation or inactivation of the aggregation receptor. AR, aggregation receptor; AF, aggregation factor; CPP, circular proteid particle; GluA,'glucuronic acid; [~

inactive AR; ~

active AR

Cell separation (via aAR) The observation which led to the discovery of aAR was that only 75'}~; of the cells had the potency to form aggregates in the presence of AF it. However, after incubation in the presence of/~-ga[actosidase (from bovine liver), the aggregation-deficient cells became susceptible to aggregation. Therefore, we had to search for a hitherto unknown macromolecule on the cell surface which prevents AR-mediated cell aggregation. This molecule (aAR) was subsequently isolated and purified tt Addition of aAR to already formed AR-mediated secondary aggregates resulted in destruction of the aggregates 11 The biological activity of aAR can be abolished by addition of /J-galactosidase. Considering the findings that the CPPs are associated with a galactosyltransferase s and that the cell membranes are associated with a ]J-galactosidase 14, we have summarized our present working models for the biological function of active aAR galactosylated aAR) and for inactive aAR (degalactosylated aAR) in Fiqure 2. Cell separation (function o[ leetin) It is known from the above mentioned results that at the cell surface of Geodia cells an aAR with a terminal galactose moiety is present. Later, an extracellular galactose-specific lectin was isolated. Subsequent biologi-

(activation of o AR )

Ce

t~_ ~Cell ~D'-1\ recognition '~J [inactivation of oAR)

Cell separation

,? iF--

+'"°"n (moskinq of o AR ~ by lecfin )

Figure 2 Cell separation and cell aggregation based upon the interrelation between an active- and an inactive antiaggregation receptor. AR, Aggregation receptor: aAR, antiaggregation receptor: CPP, circular proteid particle; Gal, galactose; c~. glucuronic acid: A, galactose; ~>--~-o active AR: inactive aAR; [ ~ : : > - ~

active aAR

V

Figure 3 Schematic illustration of the biological activity of the Geodia lectin. AR, Aggregation receptor: aAR, anti-aggregation receptor; cPP, circular proteid particle; o, glucuronic acid; •, galactose; [ ~ . - - o . ~ ,

active AR; ~ ~ ,

active aAR:

lectin (antigalactanj

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A,qyre,qation qJ'sponye cells: Werner E. G. Miiller and Peter Vaith

cal studies ~3 revealed that the activity of aAR could be abolished by preincubation with the lectin. The data are summarized diagrammatically in Fiyure 3 and demonstrate that thlz aAR (galactan) might be masked by th( lectin (anti-galactan).

Acknowledgements This work was supported in part by a research grant from Stiftung Volkswagenwerk (I/35850; W.E.G.M.). References I 2 3

Conclusions The experimental evidence summarized above demonstrates that the Geodia cydonium model system is well suited for studies devoted to the understanding of cell recognition, for the following reasons. First, the sponges are the most simple multicellular organisms, and consist of two main cell classes, germ cells and archaeocytes. The latter are believed to be 'embryonic' cells I°, which can differentiate into any other tissue cell type. These 'differentiated' functional cells seem still to have the potency to dedifferentiate and then to redifferentiate into another cell type via the archaeocyte stage. Some of the sponge cells, especially the archaeocytes, are characterized by a high motility Iv. Secondly, from the biochemical point of view the sponges are suitable systems for cell recognition studies because they can be easily dissociated into single cells which rapidly aggregate into reconstructed functional organisms in simple, defined salt solutions. Because of the obviously more primitive differentiation stage of the sponge cells, e.g. in comparison with the retina system 18,19, it was expected that only a few AFs or even only one AF, but certainly not a ~family" of them, exist in these ogganisms. Thirdly, to understand the molecular mechanisms of cell specific recognition, the rholecular components must be isolated from an almost homogeneous tissue. This means that the starting material should not be the limiting parameter, and this prerequisite is satisfied in studies using sponges.

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4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19

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