biosynthesis of frog skin mucins: cysteine-rich shuffled modules, polydispersities and genetic...

Comp. Biochem. PhysioL Vol. 105B, Nos 3/4, pp. 465-472, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd

MINI REVIEW

BIOSYNTHESIS OF FROG SKIN MUCINS: CYSTEINE-RICH SHUFFLED MODULES,

POLYDISPERSITIES AND GENETIC POLYMORPHISM

WERNER HOFFMANN* and FRANK HAUSER Max Planck-Institut ffir Psychiatrie, Abteilung Neurocbemie, Am Klopferspitz 18a,

D-82152 Martinsried, Federal Republic of Germany (Tel. 49 89-8578-3626; Fax 49 89-8578-3749)

(Received 21 December 1992; accepted 5 February 1993)

Abstract--1. Frog integumentary mucins (FIM-A. 1, FIM-B. 1 and FIM-C. 1) consist of typical threonine- rich highly O-glycosylated (semi)repetitive domains, and cysteine-rich modules, i.e. the P-domain, the short consensus repeat and a region with high similarity to the C-terminal end of von Willebrand factor (designated here CC29-motif).

2. These modules are thought to be involved in protein-protein interactions and they have been observed in a variety of extracellular proteins. In FIMs, these modules may be involved in oligomerization processes leading to an entangled mucin network.

3. Polydispersities have been detected in FIM-B. 1 and FIM-C. 1 within single individuals. Multiple transcripts are probably generated by alternative splicing of a huge array of different (semi)repetitive cassettes encoding the threonine-rich domains.

4. Furthermore, genetic polymorphism is observed between different individuals, probably due to allelic variations in the number of (semi)repetitive cassettes.

INTRODUCTION

Many epithelial surfaces, e.g. of the gastrointestinal, tracheo-bronchial and urogenital tracts, and the oral cavity, are protected against noxious influences by mucus gels. These viscous matrices consist of 95% water, with mucin-type glycoproteins represent- ing the principal structural components (Neutra and Forstner, 1987; Strous and Dekker, 1992). Also, the integument of many Amphibia, fish etc. is covered by a mucus layer which shields against dehydration, physical damage and microbial infec- tions. The latter function is often supported by a variety of antimicrobial peptides (Zasloff, 1992). Furthermore, mucin-type glycoproteins have been found in membrane-bound forms in different tumor cells (Hilkens et al., 1992) and mucin-like molecules also form characteristic components of various glues, e.g. from Drosophila salivary glands (Garfinkel et aL, 1983).

Classical biochemical investigations, as well as recent molecular approaches, generally defined mucins by their characteristic highly O-glycosylated domains (for review, see Strous and Dekker, 1992). Typically, these domains contain a (semi)repetitive protein backbone with a particularly high content of threonine or serine residues interspersed by proline residues. This reflects the importance of secondary

*To whom correspondence should be addressed.

structure for O-glycosylation by GalNAc transferase (O'Connell et al., 1991). The clustered O-linked oligosaccharides result in a stiff, extended confor- mation of mucins (Jentoft, 1990). Due to sulfation of carbohydrate moieties and terminal sialic acid residues, mucins can be considered as highly charged thread-like polymers. As a consequence, during exocytosis the mucin matrix undergoes an ion-triggered transition from a condensed to an expanded hydrated phase, caused by the mutual repulsion of the poly- anionic charges (Verdugo, 1990, 1991). This massive swelling is the biophysical basis for many general properties typical of mucins.

Up to 80% of the molecular mass of mucins consists of clustered O-linked short carbohydrate side-chains. However, there are also "naked" stretches present in the protein backbone which are devoid of O-glycosylation. Normally, these regions are sensitive to digestion with proteases. Further- more, these regions contain cysteine residues which are thought to be responsible for oligomerization of mucins via interchain disulfide bridges (Dekker and Strous, 1990). This process is thought to be important for the gaining of viscous gel-forming properties. A mainly linear assembly is thought to occur end-to- end (Carlstedt et aL, 1985; Dekker et al., 1991; Strous and Dekker, 1992), but polymerization via non- mucin "link" proteins has also been discussed (Allen, 1983). For example, a fibronectin fragment has been identified as a link protein in intestinal mucin

465

466

FIM-A.1

WERNER HOFFMANN and FRANK HAUSER

; T ? T ~t,lr,~ttTt T??? ?,r ~rT,rr ?: :;; ; : ; ~ ' / / / / ~ , IU l I I I I I I I I I I IB I I I I I I ! I ' / / / / ~ ~1

P P P P

r TTTTT. T '" ' T.T T T F * M - . . I ~ l l ] l l l l l l l l l l l l l l l l l ~ ' / / / / / ~ l UIIlllllllll ~///////////////////////////////////////~

5CR CC29

FIM -C.1

. t t t r T vY 1?l?tttt1'tT I'ff?TT?Yt?Tt? f~ ~/'////~d ~////////////~ ~'////~///7/~'////~

r

P P P P P P 100 l a I

Fig. 1. Schematic representation of FIM-A.1 (Hoffmann, 1988), and the carboxy-terminal portions of FIM-B.I (Probst et al., 1990; Probst et al., 1992) and FIM-C.1 (Hauser and Hoffmann, 1992). Cysteine-rich modules are hatched (P, SCR and CC29). Knobs represent O-linked sugar moieties in the (semi) repetitive domains whereas squares indicate potential N-glycosylation sites. The arrow indicates

the cleavage site for signal peptidase in the FIM-A. 1 precursor.

(Slomiany et al., 1991, 1992). Generally, cleavage of disulfide bonds results in the dispersion of mucin gels, reducing their viscosity. However, kinetic data favor an "entangied-network model" for mucus polymer matrices rather than rigid cross-linking among polymer chains (Verdugo, 1990).

Our knowledge concerning the full molecular structure of mucins is still very limited. This is mainly due to experimental difficulties caused by genetic polymorphism of mucins (Swallow et al., 1987), as well as their extreme length, their repetitive nature and extensive O-glycosylation. As a further attempt to understand the function of cysteine-rich modules in mucins, we investigated various integumentary mucins from Xenopus laevis which have been discovered solely by molecular cloning.

the terminal cysteine-rich regions are accessible to proteolytic degradation (lane b). In agreement with the cDNA sequence, digestion with N-glyco- peptidase F revealed that FIM-A.1 is also N-glycosylated.

Cysteine-rich modules

As shown in Fig. 1, three different types of cysteine-rich modules have been characterized. In FIM-A.1 and FIM-C.1, numerous units of P- domains (term according to Tomasetto et al., 1990) are present whereas, in FIM-B. 1, the short consensus repeat (SCR; also termed sushi structure) and a region with significant similarity to the C-terminal end of von Willebrand factor (vWF) are found. As is typical of many shuffled modules (Patthy, 1991), the

MOLECULAR ANALYSIS a b c d

So far, three frog integumentary mucins (FIMs) with different (semi)repetitive elements have been characterized. They have been designated FIM-A.1, FIM-B.1 and FIM-C.1 (Hauser et al., 1990). Each FIM represents a typical mosaic protein built up of O-giycosylated threonine-rich domains and cysteine- rich modules (Fig. 1). From lectin analysis many more FIMs as constituents of integumentary mucus from X. laevis are to be expected (Schumacher et al., in preparation). However, FIM-A.1 represents the predominant integumentary mucin (Probst et aL, 1992).

As the typical repetitive element in FIM-A.1, which is the first entirely sequenced mucin described, the module VPTTPETTT has been identified (Hoffmann, 1988; Hauser et al., 1990) whereas in FIM-B,1 the motif GESTPAPSETT appears (Probst et aL, 1990, 1992). Characteristic of FIM-C. 1 are semi-repetitive cassettes such as KATTTTP ' ITr (Hauser and Hoffmann, 1992). In Fig. 2, the existence of a protease-resistant highly O-glycosylated core is demonstrated for FIM-A.1 (lane c) whereas

2 0 5 -

1 1 6 -

9 7 -

6 6 -

4 5 -

Fig. 2. Proteinase K resistance of the highly O-glycosylated portion of FIM-A.1. Shown is a SDS-PAGE (7.5%) and subsequent Western analysis with the antiserum against the C-terminal end of FIM-A. 1 (Hauser et al., 1990; lanes a and b) or lectin analysis with Helix pomatia agglutinin (lanes c and d). Lanes a and d: mucus from X. laevis skin. Lanes b and c: after digestion of the mucus samples with proteinase K, the terminal P-domains in FIM-A. 1 are missing (lane b) resulting in a reduced molecular weight of the lectin-positive

band (lane c).

Frog integumentary mucins 467

SCR and probably most P-domains are encoded by separate exons flanked by phase 1 introns.

In analogy to epidermal growth factor (EGF) and EGF-like repeats, the P-domain also represents the basic unit for a rapidly growing family of secretory peptides with potential cell-growth modulating activity. They are predominantly expressed in the gastrointestinal tract, the pancreas or frog skin and consist of one (pS2/xP1, rITF; Jakowlew et al., 1984; Hauser and Hoffmann, 1991; Suemori et al., 1991), two (hSP/PSP/mSP, xP2; Tomasetto et al., 1990; Hauser et al., 1992) or four (xP4; Hauser and Hoffmann, 1991) such modules (homologues from different species are separated by slashes). As a hallmark, this motif of approximately 50 amino acid residues contains six highly conserved cysteine residues, a single arginine, a glycine and a tryptophan residue (Hauser and Hoffmann, 1992). The six cysteine residues form three intramolecular disulfide bridges with the preliminary assignment Cys-1/Cys-5, Cys-2/Cys-4 and Cys-3/Cys-6 (Thim, 1989). Se- quences with remarkable similarity to P-domains have also been detected in the sucrase-isomaltase complex of the small intestinal brush border membrane and in lysosomal ~-glucosidase (Tomasetto et al., 1990). Thus far, the molecular function of P-domains is unknown. Only for PSP have general similarities with EGF/urogastrone been reported, i.e. a mitogenic effect (Thim, 1989; Hoosein et al., 1989)

and an inhibitory effect on gastric acid secretion and intestinal motility (Jorgensen, 1982). Furthermore, specific receptor-binding of PSP by rat intestinal mucosa cells has been described (Frandsen et al., 1986; Frandsen, 1988).

The SCR detected in FIM-B.1 is a motif typical of many complement control factors interacting with C3b or C4b (Reid et al., 1986) and many non-complement proteins (compiled in Probst et al., 1992). This shuffled module consists of about 60 amino acid residues and the four conserved cysteines are disulfide-bonded in the pattern Cys-1/Cys-3 and Cys-2/Cys-4 (Lozier et al., 1984; Janatova et al., 1989). Strictly conserved are further a glycine and a tryptophan residue. The precise function of SCRs is currently not understood on a molecular level, but it is thought they might represent protein-binding modules.

A third cysteine-rich module has been detected at the C-terminal end of FIM-B. 1. This region has a length of 228 amino acids and shows particular similarity to part of the CI domain and the C- terminal end of vWF (Probst et al., 1990). Remark- ably, 29 out of the 30 cysteine residues in FIM-B.1 are conserved (Fig. 3). In the human vWF gene (Mancuso et al., 1989), this region starts precisely at exon 43 and further comprises exons 44 and 49-52. This module with the characteristic 29 cysteine residues, has also been detected in the C-terminal

FIM 170 GSSG~SVQAGHMWQT GT .. . . . . SGKT PR KEI SDERRVLRKPGK -- PL HNGTEYKLGA BSM 339 ~gYGPLG~KK SPGD I WTA~HK~I~TD .. . . . . . AETVD~KLKE~PSPPI~IKPEERLVKFKDND~ IA~EPRI~LFNNNDYEVGA

9 3 3 . . . . . . .

rMLP 582 ~VHEN~ET/QPGSPVYSN~QDF~qTDSMDNSTQLNVI S~THVP~- -N I ~(~SSGFELVE- .VPGEFC~- .K~QQT~I i KRPEQQy i I

vWF 166 / ~HRST I YPVGQ-FWEE(~DV~F~rDMEDAVMGLRVAQ~SQKI~- - EDS~R~FTYVL- -HEGIZ~.qG. -p,~I.pSA~EVVTGSPRGDS

12 13 i, is 16-i~ f BSM SFA .. . . . DPKNP~I SY~- --HNTGFV--AVVQDFF~I(- -QTW~AEEDRVYD- - ST~C~y" ~( . . . . . . PY~RSSSVNVTVNYNG . . . . . . . . PSM SFD .. . . . DPNNP~TY~- - -QNTGFT--AVVQ~- -QTI~IAEEDRVYD-- SKqCC~ ~( . . . . . . SS~KPSPVNVTVRYNG . . . . . . . . rMLP LKPGEIQKNPNDR~FF~IKINNQLIS-SVSNIIF~I)FDPSD~PGSITYM..PN(~CC~ IIHNPNNTVP~AIPVMKEISYNG . . . . . . . . MUC2 LKPGD~SDPKNN~FFg~VKIHNQLIS-SVSNIIFP~FDASI~IPGSITFM--PN(~CC~(]_._____ ~TPRNETRVP~STVPVTTEVSYAG . . . . . . . . vWF QS~DI~IDTHF~KVNERGEYFWEKRVTGL~PFDEHK~I.AEC~? I MK I pGl~_.~Dl ~E .. . . . EPE~WDITARLQYVKVGS . . . . . . .

411

20 22 23-25 26 2 28 29

~I(KKVE~--F ~KT I K~DYD IFQLKNSF~CC~EENYE~E I D~D~D~GT I pYR~H ~H S~ILD. I~QQSMTSTV S

~(SEVEVDI H'~ ~(~kSKAI~'~j~ I D I NDVOD(~I~_~PTRTEPMQV.~::~I,~gTI~SVVY H EVLNAMI K~ISPRI(~;K $I

Fig. 3. Similarity of the cysteine-rich C-terminal domains (CC29) of various mucins with von Willebrand factor. FIM, FIM-B.1 (Probst et al., 1990); BSM, bovine submaxillary mucin (Bhargava et al., 1990); PSM, porcine submaxillary mucin (Eekhardt et al., 1991); rMLP, rat intestinal mucin-like peptide (Xu et al., 1992a); human MUC2 (Gum et al., 1992); vWF, human von Willebrand factor (Titani et al., 1986). Gaps are introduced to maximize homology; amino acid residues identical at least in all mucins (FIM, BSM, PSM, rMLP, MUC2) are enclosed in boxes. The conserved 29 cysteine residues are numbered. Triangles mark the corresponding positions where introns are located in the vWF gene (Mancuso et al.,

1989).

FIN BSM PSM rMLP MUC2 vWF

468 WEANER HOFFMANN and FRANK HAUSER

regions of bovine and porcine submaxillary mucins (Bhargava et aL, 1990; Eckhardt et al., 1991), of a rat intestinal mucin-like peptide (Xu et al., 1992a) and its human homologue MUC2 (Xu et al., 1992b; Gum et al., 1992). The spacing of the 29 cysteine residues in this cysteine-rich C-terminal (CC29) module is variable to some extent (Fig. 3). Most highly conserved is the region between Cys-21 and Cys-28. So far, the molecular function of the CC29-domain in mucins is not known. However, part of the corresponding region in vWF, i.e. the C-terminal end, including Cys-12 to Cys-29, has been shown to be sufficient for dimerization via intermolecular disulfide bridges (Voorberg et al., 1991). This process is independent of other domains, such as domains C1 or C2. Consequently, one could speculate that a similar process occurs in mucins. This hypothesis is currently the subject of detailed investigations. Interestingly, a shortened and slightly different version of this CC29-motif starting at Cys-20 has recently been found at the C-terminal end in various extracellular proteins (e.g. the slit protein and the Norrie gene product); here, also a role for this motif in protein-protein interactions has been postulated (Rothberg and Artavanis-Tsakonas, 1992) and mutations in the corresponding region of the Norrie gene are thought to represent the molecular basis of this disease (Meindl et al., 1992). Furthermore, based on a previous report (Hunt and Barker, 1987), a carboxy-terminal truncated version of the CC29-domain (containing Cys-1 to Cys-10) can be defined in thrombospondin and the amino-terminal pro-peptide of the collagen precursor (Fig. 4). In procollagen this region is encoded by a separate exon (Chu et al., 1984) whereas in the thrombospondin gene it is divided between two exons (Bornstein et al., 1990).

Polydispersities by alternative splicing

Starting from single individuals, a population of polydisperse mRNAs for FIM-B.1 and FIM-C.1 is always observed (Hoffmann, 1988; Hauser and Hoffmann, 1992). In contrast, the transcript length for FIM-A.I seems to be more uniform (Hoffmann, 1988). These mRNAs probably originate from two genes only and differ by various deletions/ insertions in the (semi)repetitive threonine-rich

domains. The mosaic pattern of these deletions/ insertions led to the proposal of a cassette model which explains the highly variable (semi repetitive parts in FIM-B.1 and FIM-C.I by alternative splicing. In agreement with this model, repetitive elements are encoded by separate exons in FIM-B. 1 (Probst et al., 1992). This highly complex structure of the FIM-B.1 (and probably also the FIM-C.1) gene is in contrast to the structure of other mucin genes where the repetitive domain is not interrupted by introns, e.g. MUCI (Spicer et al., 1991), MUC2 (Toribara et aL, 1991) or Drosophila glue proteins (Garfinkel et al., 1983).

Genetic polymorphism

As is particularly well-documented for MUC1 (Swallow et al., 1987), all FIMs investigated thus far are genetically polymorphic, i.e. each individual can be recognized by its unique pattern of FIMs (Hauser et al., 1990; Probst et al., 1992; Hauser and Hoffmann, 1992). Figure 5 illustrates the polymorphism for FIM-A. 1. This polymorphic appearance is typical of many repetitive proteins such as surface proteins of malaria parasites (Mackay et al., 1985) and proline-rich proteins from salivary glands (Azen et al., 1984). Most commonly, polymorphism is due to a variable number of tandem repeats, e.g. between 20 and >100 in MUC1 (Lancaster et al., 1990). However, in order to maintain the at least semi- repetitive nature of these elements, a gene conver- sion mechanism seems likely, preventing a common genetic drift.

BIOSYNTHESIS

X. laevis skin secretions are produced by two different types of glands (Engelmann, 1872; Spannhof, 1953, 1954; Fox, 1986). In response to irritation, regenerative granular glands (Flucher et al., 1986) secrete, via a holocrine mechanism, biogenic amines (Erspamer, 1971), a variety of hormone-like and antimicrobial peptides (Bevins and Zasloff, 1990; Gibson, 1991) and a cytoplasmic lectin (Bols et al., 1986; Marschal et al., 1992).

In contrast, probably due to missing direct innervation (Sj/Sberg and Flock, 1976; Sjfberg, 1977), merocrine mucous glands continuously release their

FIM-B.I

B~

P~ P~Z(Z) mTSP1 mTSP2

1 2 3 4 5 6 7 89. 10 171 ~CGSS~ESVQAGHI~-TI~D~NGTSGKT(~RI~EKE I I~SDERRVLRKPGKSF~-'GY~EP • •. 340 ~fGPL~KKSPGD I~I~'-AI~I~D-AETVI~LKEF~SPPI~PEERLVKFKDNDTT I AY~I~EP • • • 934 ~I4GPL~EKSPGD~"-AI~I~E-AKTVI~PKIE~PSPPI~TGERL I KFKANDTT I GI~K • •.

18 ~1/-QI~RYHDRD~PEI~ ~ D N - -GKVIL.~V I~I~I3ETKI~GAEV . . . . . PEGTC~--PVI~PD... 318 ~F -HN~VQYKNNEE~rVDS~TE~I~N- - SVT I~(KVS~P INP-~SNATV . . . . . PDGT(~--PR~P... 318 ]~/-QE~ I FAENE]~/VDS~TllC~K- -FKTV~4QI T~-SPA]Z~ANPSF . . . . . VEGE~-Pg~SH...

Fig. 4. Similarity of mucins with procollagen and thrombospondin. Shown is the region between Cys-1 and Cys-10 of the CC29-domain. FIM-B.1 (Probst et al., 1990); BSM, bovine submaxillary mucin (Bhargava et al., 1990); PSM, porcine submaxillary mucin (Eckhardt et aL, 1991); Pgl(I), human pro~, l(I)-collagen (Chu et al., 1984); mTSP1 and mTSP2, mouse thrombospondin 1 and 2 (Bornstein et aL, 1991). Gaps are introduced to maximize homology; highly conserved amino acid residues are enclosed

in boxes.

Frog integumentary mucins

a b c d • f

469

2 0 5 -

1 1 6 -

9 7 - 6 6 -

4 5 -

Fig. 5. Genetic polymorphism of FIM-A.I. Shown is an SDS-PAGE (7.5%) of ,I". laev/s skin secretions from six different individuals and subsequent Western analysis with the antiserum against the C-terminal

end of FIM-A.I (Hauser et aL, 1990).

products (Spannhof, 1953, 1954). Also, adrenergic stimulation has been reported to induce secretion of an ionic outflow consisting of Na + and CI- (Skoglund and Sj6berg, 1977a,b). Histochemically, mucous glands differ considerably in their poly- saceharide content and their lectin-binding pattern from granular glands (Spannhof, 1953, 1954; Danguy and Genten, 1989, 1990). In X. laevis, mucous glands appear during metamorphosis about 1 or 2 days behind the granular glands. Surprisingly, muco- polysaccharides are detectable even in premeta- morphic skin (Shih and Vanable, 1975). Two different types of secretory cells lining the mature mucous gland have been described: ordinary mucous cells and up to four cone cells at the proximal pole of the gland (Spannhof, 1953, 1954). The presence of more than one secretory cell type in dermal mucous glands has been demonstrated, also, in Rana fuscigula (Els and Henneberg, 1990).

By immunohistochemical staining as well as in situ hybridization histochemistry, synthesis and storage of frog integumentary mucins have been localized exclusively to mucous glands (Hauser et al., 1990; Probst et al., 1990). As shown in Fig. 6, only ordinary mucous cells store FIM-A.1 within secretory granules; cone cells at the proximal pole of the gland are devoid of this mucin. In contrast, FIM-B.I and FIM-C.1 seem to be enriched within cone cells (Schumacher et al., in preparation). Based on this result, cone cells can be considered as specialized cells rather than degenerating ordinary mucous cells as proposed previously (Spannhof, 1953, 1954). The massive storage of FIM-A. 1 within granules would be an indication of secretion via the regulated pathway. However, the stimulus responsible for this more or less continuous release of FIMs is not completely understood, although a fl-adrenerglc mechanism apparently triggers exocytosis in mucous glands whereas granular glands respond immediately to

~t-adrenergic stimulation (Benson and Hadley, 1969; Skoglund and Sjtberg, 1977a,b; Mills, 1985).

PERSPECTIVES

The presence of the numerous cysteine-rich modules is a somewhat surprising feature of FIMs. These domains may be important for various biologi- cal functions. For example, the SCR might interfere directly with the adhesion process of microbia thus protecting the skin from infection (Probst et al., 1992).

Alternatively, we favor the idea that cysteine-rich domains would be ideal candidates to be involved in the oligomerization of mucins. Based on this assump- tion, the three different motifs could be distinguished by their capability to form intermolecular disulfide bridges. Due to the homology with vWF, the CC29- module might form such cross-links leading to dimerization. In contrast, P-domains and the SCR are not hkely to be candidates for covalent interactions. They preferentially form intramolecular disulfide bonds and non-covalent interactions with various proteins can be expected. Also, a lectin-like binding of cysteine-rich modules to carbohydrate epitopes seems reasonable. Interestingly, the hypothesis of a lectin-bond mediated polymerization of mucins has been proposed in the past (Silberberg, 1987). In particular, P-domains would be possible candidates. Interestingly, secretion of P-domain peptides occurs in mucin-producing cells (e.g. surface mucous cells and goblet cells of the gastrointestinal tract) or is associated with tumors showing mucinous differen- tiation. Thus, P-domain peptides might also function as putative link proteins.

The non-covalent interaction of cysteine-rich modules in mucins has been proposed in the past (Roberts, 1976) and the presence of these low energy intermolecular bonds would fit with an "entangled-

CBP(B) IO~-3/~-D

470 WERNER HOFFMANN and FRANK HAUSER

Fig. 6. Immunohistochemical analysis of a single mucus gland from X. laevis skin. After treatment with the anti-FIM-A.1 antiserum (Hauser et al., 1990), immunofluorescence can be detected on the surface of the epidermis, in the lumen of the gland and within granules of the ordinary mucous cells but not within

the two cone ceils at the proximal pole of the gland. Scale bar = 25/~m.

network model" (Verdugo, 1990). The existence of covalent branch points via internal cysteine-rich modules (i.e. the SCR in FIM-B. 1 and P-domains in FIM-C.1) would not easily be compatible with this model. In contrast, a non-covalent branching of the oligomer may still allow an entangled mucin network. So far, our knowledge concerning the molecular structure of internal cysteine-rich modules in mucins is limited. Complete internal regions, other than P-domains and the SCR in FIMs, have been characterized only in MUC2 (Gum et al., 1992). The search for eysteine-rich modules in mucins should bring new insights into the understanding of their oligomeriza-

tion and, in consequence, their diverse rheological properties.

Acknowledgements--We thank Dr R. A. Hughes for criti- cally reading the manuscript and Prof. U. Schumacher (Southampton) for stimulating discussions. Part of this work has been supported by grants from the "Deutsche Forschungsgemeinschaft" and the "Schilling Stiftung".

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