Chapter 11 Human mucosal mucins in diseases

J. Montreuil, J I G . Vliegenthart and H. Schachter (Eds.), Glycoproteins and Disease 0 1996 Elsevier Science B.V. All rights reserved CHAPTER 1 1

Human mucosal mucins in diseases Philippe Roussel and Genevitve Lamblin INSERM I/ 377, place de Verdun, 59045 Lille Cedex, France

Abbreviations CF

cystic fibrosis

LeX

Lewis x

CFTR

cystic fibrosis transmembrane regulator

PEM

polymorphic epithelial much

HMGF

human milk fat globule

PUM

polymorphic urinary mucin

HPA

Helix pomatia agglutinin

vWF

von Willebrand factor

Lea

Lewis a

VNTR

variable number of tandem repeats

Leb

Lewis b

1. Introduction: “mucin”, an elastic word Mucus covers the surface of many epithelia (respiratory, digestive, genito-urinary, ocular) and represents an interface between the environment and the “milieu interieur”. Mucus is a mixture of several secretions and has rheological properties (elasticity and viscosity). It protects the underlying epithelium against various types of aggression. Among the different families of molecules which are responsible for these protective properties (secretory antibodies, antiproteases, antimicrobial peptides such as lysozyme or defensins, iron-chelating proteins), mucins are the main constituents of mucus. These very complex 0-glycoproteins play a major role in the defence of the mucosae. The word m u c h is an ancestor in the field of glycoconjugates. For more than a century the concept of mucin, or mucus glycoprotein, was associated with material secreted by mucosae. Mucins (including blood group substances) were probably the first type of compounds to be clearly recognized as glycoproteins. However the ancestor is dynamic and its definition has a tendency to be unstable. For a while, there was some confusion between mucus and mucin and the term “mucus glycoprotein” was preferred to mucins. However the word has overcome this disgrace and the definition of mucins has been based on their chemical composition (from 50 to 80% carbohydrate) and molecular mass (from several hundred to several thousands kDa). Mucins are glycoproteins containing from one to several hundred carbohydrate chains attached to the peptide by 0-glycosidic linkages between N-acetylgalactosamine and an hydroxylated amino acid (serine or threonine). Frequently the carbohydrate chains are clustered in highly glycosylated domains and the usual representation of mucins is that of a “bottle-brush” (Fig. 1). In most human mucins, the carbohydrate/peptide ratio (by weight) is more than three. Apomucins, the peptide part of the mucins, have a high proportion of hydroxylated amino acid (serine + threonine). They are encoded by different mucin genes (MUC genes) and the sequencing of several cDNAs from MUC genes 351

352

carbohydrate chains

naked region

highly glycosylated domains Fig. 1. Schematic representation of a much molecule with naked regions and highly glycosylated domains (giving mucin glycopeptides after proteolysis).

has shown “tandem repeats” with high proportions of hydroxylated amino acids. These tandem repeats are frequently but not always present. Typical human mucins contain fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. They may also contain sulfate and small quantities of mannose. The physical polydispersity and several other lines of evidence clearly indicate that, in a given mucosa or in the secretion of a given exocrine gland, there is a very large family of mucin molecules differing from each other, at the peptide as well as at the carbohydrate levels. In this definition, mucins are also linked to the specialized cells responsible for their biosynthesis, goblet cells and mucous cells in mucosae or exocrine glands. However the meaning of the word mucin has recently been extended to membranebound components found in many cancer cells of epithelial origin and called “mucinlike”, because they have highly glycosylated domains and sometimes tandem repeats in their peptide part [l-31. Interestingly, some of these “mucin-like” molecules can be shed, rather than secreted, from cancer cells, and sometimes also from normal epithelial cells, although to a low extent. It has been suggested to designate these “mucin-like” substances as epithelial mucins, as opposed to gel-forming mucins [4];however both types of glycoproteins have an epithelial origin and, moreover, some secreted mucins do not form gels. More recently the confusion increased since other membrane-bound glycoproteins found in leukocytes and endothelial cells, which are receptors for selectins have also been designated as “mucins” [5]. In the present review, we will limit the discussion primarily to mucins secreted by mucosae or exocrine glands of human origin, and we will define them as mucosal or secreted mucins. The reader interested in “mucin-like” substances will find several excellent reviews on this topic in the literature [ 1-3,6]. Several reviews on mucins in general [7] or on mucins from different mucosae, i.e., nasal [S], gastrointestinal [9,10], respiratory [ 11,121, and salivary [ 131 have also been published. The first part of this chapter will describe the evidence which suggests that human mucosal mucins form broad families of different glycoproteins stemming from two events, the expression of different mucin genes to form multiple apomucins, followed by a wide variety of post-translational phenomena, mainly 0-glycosylation, leading to carbohydrate chains with a vast microheterogeneity. In order to illustrate the differences between “mucosal mucins” and “mucin-like” substances, current knowledge concerning episialin,

353

a mucin-like glycoprotein, encoded by a gene called MUCl, will be briefly summarized. Finally, the biological significance of the diversity of mucosal mucins will be discussed, as well as what is known about their pathophysiological modifications.

2. General properties of human mucosal mucins 2.1. Cellular origins of human secreted mucins

In human mucosae and exocrine glands (mucous or sero-mucous glands), two types of cells are involved in the synthesis and secretion of mucins [14,15], the goblet cells and the mucous cells. Cells with a similar function are also found in gland ducts, including sweat ducts [ 161. The goblet cell found in different epithelia (gastro-intestinal, respiratory, cervical, . . . ) is the typical mucin synthesizing cell [9,17]. At the basal part, it has a nucleus surrounded by rough endoplasmic reticulum and an apical part is filled with mucin granules intensely stained by Schiff reagent after periodate oxidation. The Golgi apparatus is located between these two cell compartments. The cells which form the mucous glands in the various submucosae or in mucous exocrine glands have a similar goblet shape. The mucin peptides are thought to be translated in the rough endoplasmic reticulum as apomucins and most of the glycosylation process occurs in the Golgi apparatus, which produces mucus granules that accumulate at the apical part of the mucin secreting cells prior to secretion. These different cells may differ in their staining intensity with different dyes, such as Schiff-periodate, as well as in their affinity for different lectins [18,19] or antibodies [20]. When using electron microscopy with specially adapted techniques equivalent to Schiffperiodate, differences in the density of mucin granules may be observed from one cell to the other, sometimes from one granule to the other within the same cell [9,21], already suggesting a diversity of the mucins synthesized even by an individual cell.

2.2. Physico-chemical properties of human mucins Mucus and mucins have rheological properties (viscosity, elasticity) which are important for their physiological function (for instance, the efficiency of the mucociliary escalator in the airways). However these properties represent a major difficulty when working with mucins. Mucus has to be solubilized before the purification of mucins. For this purpose, various mucolytic procedures have been used but some of them, such as proteolytic enzymes or reducing agents, obviously produce some degradation of the mucin molecules. Mild agitation in dissociating agents, or after dilution with water, leads to disentanglement of mucin molecules which are then ready for chemical or physical analyses [22,23]. During exocytosis, mucins undergo considerable swelling suggesting that their rheological properties may be regulated by the concomitant movements of water, ions and soluble proteins [24]. Based on their peptide and carbohydrate composition and on their susceptibility to alkali, the usual representation of mucins is that of a “bottle-brush” with many

354

Table 1 Molecular Mass of different human mucins (x 1O6 Da) Mucins Gastric Cervical Respiratory normal bronchitic

Sedimentation equilibrium 1.8-2.2

3.3-1.1 1

asthmatic

CF

I .8

Ref.

Light scattering

Ref.

28

29 30 22

I1

32

14-16

26

3.5 15-20

9.3 3.8

31 34 33 31

carbohydrate chains attached to serine and threonine residues of the mucin peptide. Mucins may contain two types of domains, “highly glycosylated” regions, and “naked” domains[25] (Fig. I). Proteases degrade the naked domains, which are more or less devoid of carbohydrate chains, leaving “highly glycosylated” regions resistant to proteolysis, designated as mucin glycopeptides [23] or T-domains [26,27]. The estimated molecular mass of most human mucins is still a matter of debate (Table 1). There are large differences according to the method used: in the range 1l o x 1000kDa with sedimentation equilibrium [22,28-301, and 3-2Ox 1000 kDa with light scattering [26,3 1-34]. However there is general agreement over the large polydispersity of these molecules. A major advance in the understanding of mucin conformation occurred with the introduction of electron microscopy for studying mucin molecules [351. Human mucins appear as polydisperse, linear and apparently flexible threads [36-391. However, for the mucins secreted by a given tissue, there are some discrepancies between different laboratories with regard to the width of distribution. In the data reported for respiratory mucins by Slayter et al. [36] and Rose et al. [37], the distribution of the filaments ranged between 200-300nm and about 1500nm. Larger species up to 5000nm have been described by Sheehan et al. [39]. Frequently, electron microscopy also shows aggregates and it is difficult to firmly establish whether the longest filaments correspond to individual mucin molecules[26,39] or to tangled units. Mucins also have lipidbinding [30] and hydrophobic properties which can contribute to their polymeric structure through noncovalent interactions [40]. Mucin glycopeptides obtained by proteolysis of purified respiratory mucins [26] or by direct reduction of respiratory mucus [41] appear to be polydisperse, although as shorter rods with a distribution of sizes ranging from 50 to 250nm. For many mucins, reducing agents act on the longer species to produce shorter species [27,41]. From such experiments, it was concluded that mucin subunits were

355

S

\

\

S

Fig. 2. Oligomeric organization of mucins such that subunits are linked end to end.

linked by disulfide bridges end to end (Fig. 2). However the subunits obtained from the mucins of a given mucosa are still heterogeneous and the reasons for this heterogeneity are unclear (multiple types of subunits due to multiple genes?; heterogeneity in the transcripts?; post-translational proteolysis?). Moreover, it is still unknown if the reducing agents are acting on inter- or intra-molecular disulfide bridges. Silberberg and Meyer [42] have hypothesized that intramolecular disulfide bridges might create domains within a subunit allowing non-covalent interactions with another subunit. In other mucins, “link proteins” covalently attached to mucin subunits have been reported [43]. As a matter of fact, such link proteins appear to be carboxy-terminal and poorly glycosylated fragments of mucins [44]. 2.3. The wide diversity of human apomucins 2.3. I . Human apomucin polydispersity Since human mucins appear as polydisperse glycoproteins, even when collected directly from healthy areas of human mucosae, several laboratories have designed experiments to characterize the size of the apomucins, or peptide precursors. In the case of human respiratory mucins, Perini et al. [45] have characterized the apomucin precursors in the rough endoplasmic reticulum, before glycosylation in the Golgi apparatus. They prepared antibodies against deglycosylated products of “highly glycosylated” regions isolated from human respiratory mucins [46,47]. These antibodies, which recognized uncovered mucin peptides, or apomucins, were used to immunoprecipitate radiolabelled mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling experiments with [3H] threonine. They demonstrated the existence of a broad population of peptide precursors in the range of 200-400 kDa [48]. The same antibodies were also used to characterize the respiratory mucin precursors obtained during in vitro translation experiments of mRNAs purified from human tracheobronchial mucosa [45]; these precursors appeared as a polydisperse population of peptides in the range of 100 to more than 400 kDa. However Klomp et al. [49,50], working on human gastric and gall-bladder mucins, have observed more discrete bands at 470 kDa and 500 kDa, respectively. 2.3.2. Mucin cDNAs and chromosomal localization of human mucin genes Setting up deglycosylation procedures (HF, TMSF) for mucins [5 I] has been extremely useful to prepare antibodies which have been used in the cloning of mucin genes. How can we explain the much diversity at the peptide level? To answer this question, cDNA libraries from different mucosae or exocrine glands (reviews in refs. [52,53]) have been constructed in expression vectors and screened with antisera directed against apomucin sequences prepared by mucin deglycosylation [54,55].

356

Table 2 Mucin genes Gene

Chromosomal location

MUCl

1 q2 1-q24 llp15.5

MUCZ MUC3 MUC4 MUCSAC MUC5B MUC6 MUC7

7q22 3q29

llp15.5 1 lp15.5 llp15.5 4

Size of tandem repeat (number of amino acids)

Remarks

20 23 17 16 8

Degenerate tandem repeat I 69 23

6 repeats

Within the past few years, a whole range of cDNA sequences from different origins have been reported (respiratory, intestinal, gastric, salivary, . . .). Most of them contain numerous sequences of amino acids that are repeated in tandems of various lengths [54, 56-58] (Table 2). These “tandem repeats” are rich in serine and threonine residues and correspond to the most glycosylated part of the mature mucins. Such repeats are found in gastric, intestinal, salivary and respiratory human mucins. The length of the repeats ranges from 8 to 169 amino acids. In some respiratory apomucins, there may be imperfect repeats [59] (Table 2). In other respiratory mucins, there are no repeats at the peptide level but degenerate repeats at the DNA level, responsible for apomucins containing alternating hydrophilic and hydrophobic domains [60] (Table 2). The genes encoding for human secreted mucins are located on different chromosomes (Table 2). The human intestinal mucin genes, MUCZ and MUC3, were mapped to the p15 band of chromosome 11 [61,62] and on chromosome 7 [57], respectively. For human tracheo-bronchial mucins, several genes have been identified on chromosomes 11 in p15 (MUCSAC and MUC5B) [63] and 3 in q29 (MUC4) [58]. Most of the mucin cDNA sequences described so far are partial, except for two full length cDNAs which have been reported in the cases of intestinal mucins (MUC2) [64,65] and salivary mucins (MUC7) [66].

2.3.3. MUC2 The complete MUC2 cDNA that has been sequenced corresponds to an apomucin containing more than 5100 amino acids [64,65] (Fig. 3). This MUC2 apomucin has a signal sequence and two central regions rich in serine and threonine which are potential sites of 0-glycosylation: a domain of 347 residues organized in irregular ThrISerlPro repeats, and a large domain containing the tandem repeats of 23 amino acids. The remaining part of this apomucin is rich in cysteine and consists of four repetitive elements having a high degree of similarity with the D domains of the prepro-von Willebrand factor, three on the amino-terminal side and one on the carboxyterminal side (Fig. 3). These domains contain several potential sites of N-glycosylation. In vWF, the D domains are involved in the formation of disulfide-linked polymers.

357

Fig. 3. Comparison of MUC2 apomucin and prepro-von Willebrand factor (prepro-vWF). Regions homologous to the D domains of the prepro-vWF are found on both extremities of MUC2 [64,65].Two repetitive regions are nch in potential 0-glycosylation sites, the TSP-rich region and the region made of perfect tandem repeats.

There is a carboxy-terminal region to carboxy-terminal region dimerization followed by amino-terminal region oligomerization. This process is autocatalytic and depends on two sets of vicinal cysteine residues with the sequence Cys-Gly-Leu-Cys [67]. These conserved domains and the presence of an identical Cys-Gly-Leu-Cys sequence in MUC2 apomucin suggest that they might have similar functions. Allelic variants of MUC2 with a variable number of tandem repeats from 51 to 115 have been found, with different ethnic distributions [68]. 2.3.4. MUC7 MUC7 encodes for low-molecular-weight human salivary mucin, MG2, which contains 377 amino acid residues [66]. The first 20 N-terminal residues are very hydrophobic. MG2 contains a central part made of six tandem repeats of 23 amino acids rich in proline and hydroxylated amino acids, and having approximately 9&100 0-linked chains. On both sides of this region are unique sequences containing 5 N-glycosylation sites and all the cysteine residues. So far, MG2 has no sequence homology with any other mucin.

2.3.5. The MUCS and MUC6 genes In the case of human airway mucins, different types of sequences have been found corresponding to different genes.

358

Repetitive sequences of 24 base pairs, responsible for the synthesis of 8 amino acids tandem repeats [54], have been localized on chromosome I 1 (MUC5AC) [63]. This gene also contains additional repeats of 5 amino acids [69]. The 8 amino acids tandem repeat is interrupted several times by a consensus cysteine-rich subdomain of 130 amino acids containing 10 cysteine residues [70]. Such a domain is also found twice, in the central part of MUC2, between D3 and the TSP domain, and between the two repetitive domains (Fig. 3), raising the question of a common ancestor to these two mucin genes localized on chromosome 11. The involvement of these central cysteine residues in crosslinking mucin subunits has been hypothesized [70]: if this was the case, one would have to reconsider the mucin model deduced from electron microscopic studies, where mucin subunits are attached end to end (Fig. 2). A degenerate 87-base-pair tandem repetitive sequence creating hydrophilicihydrophobic alternating domains has also been characterized on chromosome 11 and corresponds to a different gene, MUCSB [60]. This gene has been recently isolated: it spans approximately 35 kb of genomic DNA, has a very large central exon (10kb) and contains 100 87-base-pair repeats, encoding for a stretch of more than 3000 amino acids composed of alternate hydrophilic and hydrophobic domains [Aubert, personal communication]. A mucin cDNA corresponding to another gene, MUC6, has been found in human stomach and gall bladder. This cDNA contains 169 amino acid repeats [71]. Like MUC2, MUC5AC and MUCSB, MUC6 is also localized on chromosome 11. Therefore the 1lp15 region contains a cluster of at least four mucin genes, which raises interesting questions with regard to their expression and regulation.

2.3.6. MUC4 A mucin cDNA, containing 48 base-pair tandem repeats responsible for the synthesis of 16 amino acids has been isolated from a human airways library [58]. The gene, MUC4, has been localized on chromosome 3. It is expressed in different tissues especially the endocervix [721. 2.3.7. Other mucin genes Another cDNA has been cloned from an expression library prepared from normal human tracheal mRNA [59]. This cDNA has imperfect 41 nucleotide tandem repeats and corresponds to a new human bronchial mucin gene localized on chromosome 12 [Sachdev, personal communication]. More recently another cDNA corresponding to salivary mucin MG1 has been cloned from a sublingual cDNA library [73]. 2.3.8. Mucin gene transcripts In most instances, mucin probes have hybridized with mucin mRNAs extracted from mucosae or glands as very polydisperse signals characterized in electrophoresis as large smears [57-59,62,66,69,7 1,741. Nevertheless, MUC2 gene transcripts are identical in the intestine and trachea [75]. For unknown reasons, the polydispersity of the message may not be observed in the case of adenocarcinoma cell lines, which may express discrete bands [76].

359

2.3.9. Tissue expression and regulation of mucin genes Aside from the heterogeneity of the RNA messages coding for mucins in a given mucosa, additional complexity results from polymorphisms leading to sequence differences between individuals [58,61]. The cellular and tissue expression of mucin genes has been studied with different techniques: Northern blots on whole tissue, in situ hybridization using mucin oligonucleotide probes, and immunochemistry using specific anti-apomucin antibodies (for a review see ref. [77]). These different approaches give complementary information. However, the picture of the expression of mucin genes is not yet complete for the different mucous or goblet cells (Table 3). Table 3 Expression of mucin genes in normal mucous or goblet cells

MUCI Tracheahronch Nasal Salivary Salivary ducts Oesophagus Stomach

o/+

++

+ + + +

+ o/+ +

+ Gall bladder

MUC2 MUC3 MUC4 MUCSB MWCSAC

+

+

+/o

+ +++

Jejunum

* ++

Ileum

+

+ +++ + ++

Colon

Pancreas duct Breast Endometrium Endocervix Ovarian

+ + + + ++ ++

+

++ +++ +

++

++ ++

+

+

* +

+ ++

++ 0

+

+ +

++

+

++

++ +

0

t++

++ +

+

0

++ 0 +t

++

++

0

0

0

+

0

0

0

++ 0 +t

++ + +

MUC6 MUC7 Refs.

++ + t k

ft

+

+/0

0

+

+

+t

I

++

69,74,80,82 69 66,82 82 7 I ,82 82 71 83 80 82 83 71 82 80 83 71 82 80 83 71 82 80 81 83 82 84 82 82 72,80 85

360

Within a given tissue, there may be a limited expression of different mucin genes, suggesting a tissue-specific regulation of the expression for some human mucin genes (Table 3). Moreover there are also variations according to differentiation of the tissue [78]. There are some hints that mucin genes might be expressed in epithelial cells other than goblet and mucous cells. MUC3 may be expressed in absorptive cells in addition to intestinal goblet cells [79] and MUC4 may be expressed in ciliated cells [80]. The m u c h secreting cells of small and large intestine mainly express MUC2 and MUC3 [80-821. MUC4 is also expressed at a lower level [80,81]. MUCSAC and MUC6 are the genes predominantly expressed in the stomach, where MUC3 and MUC4 are also expressed, and MUC2 in the antrum [71,80]. Using antibodies, Carrato et al. [83] have also found expression of MUCSB, whereas Audie et al. [80] using in situ hybridization did not observe any signal with a MUCSB probe. The respiratory tree has a wide expression of mucin genes: MUCSAC [69,80] and also, to a lower extent, MUC5B, MUC4 and MUCZ [74,80]. The endocervix expresses MUC2, MUC4, MUCSAC and MUCSB [72]. MUC2 may be weakly expressed in pancreatic [84] and ovarian cells [85]. Cancer cell lines may express several mucin genes simultaneously [76,86,87]. Finally, very little is known about the mechanisms of regulation. In tracheobronchial epithelial cells, the expression of MUC2 is down-regulated by vitamin A [88] and upregulated by TNF-a[89]. In the endocervix, the expression of MUC4 seems to be increased during the luteal phase [72]. In summary, (i) there is a growing number of cloned mucin genes, (ii) for unknown reasons, most mucin mRNAs are polydisperse, (iii) there is some evidence that apomucin heterogeneity is present prior to glycosylation, and (iv) secreted mucins appear as a wide range of filaments. This raises the question: “Is there a much molecule?’

2.4. The diversity of post-translational modiJications 2.4.1. The wide diversity of 0-glycans Most mucin carbohydrate chains are joined to the apomucin through N-acetylgalactosamine in a-0-glycosidic linkages to the hydroxyl oxygen of serine or threonine [90]: these linkages are alkali-labile. Besides N -acetylgalactosamine, fucose, galactose, N-acetylglucosamine and sialic acids are also found in mucins (Fig. 4). In addition, human mucins may contain sulfate groups and a small amount of mannose [36]. They do not contain uronic acids. From the five types of monosaccharide residues commonly found in human mucins, the biosynthetic process leads to a wide spectrum of oligosaccharide structures, varying in composition, length, branching and acidity [91-1101. This broad diversity has been a major obstacle for the structural elucidation of the carbohydrate chains of several human mucins. For example, 88 different chains have been isolated from the respiratory mucins of a single individual [ 11 1-1 161 and airway mucins probably contain several hundreds of different carbohydrate chains [ 1 171. Hounsell and Feizi [98] have identified three regions in a carbohydrate chain (Fig. 4). In fact these three parts correspond to a series of glycosylation reactions. The carbohydrate

361

0-Glycosylation SA-G

I

Gn-

I

I I

S

Gn

G

G

G

Gn

I

I

B 7 GaN 0

I

I

I

I

\/"

Gn

F

3

I

periphery backbone

GaN 0

I

Fig. 4. Schematic representation of 0-glycans, i.e. carbohydrate chains 0-glycosidically linked to human respiratory peptide by linkages involving N-acetylgalactosamine (GaN) and hydroxyamino acid [senne (Ser) or threonine (Thr)]. Each 0-glycan can be described with a core, a backbone and a periphery. There may be a few N-glycans. G, galactose; Gn, N-acetylglucosamine; M, mannose; SA, sialic acid; S, sulfate.

core is formed by the first glycosylation reactions; then the backbone is formed and, finally, the periphery. 2.4.2. Carbohydrate-peptide linkage and cores of 0-glycans The only structural element shared by all mucin 0-glycans is the GalNAc linked to the peptide. Much oligosaccharide synthesis is initiated by the action of very specific enzymes, UDP-GalNAc-polypeptide-a-h'-acetylgalactosaminyltransferases, on the apomucins [ 1 181. These enzymes are probably localized in the cis-Golgi. The linkage GalNAc and the sugar(s) directly attached to it constitute the "core" region of the mucin oligosaccharides [98] (Table 4). This GalNAc can be substituted on the hydroxyl of C3 either by a Gal(P1-3) or a GlcNAc(P1-3) [119] to give core 1 and core 3, respectively [98,120]. Addition of GlcNAc in (P 1-6) linkage to core 1 and core 3 produces two other cores, core 2 and 4 (Fig. 5). Three other cores have been described in human mucins: cores 5 and 6, in meconium [102,121], where the GalNAc is substituted by a GalNac(a1-3) (core 5) or by a GlcNAc(P1-6) (core 6), and core 8, where the GalNAc is substituted at C3 by a Gal(a1-3) [122] (Fig. 5). So far no core 7 (GalNAc(a14)GalNac) has been detected in human mucins. The GalNAc residue of cores 1, 3, 5 and 8 can also be substituted by an N-acetylneuraminic acid in a 2 4 linkage. Different types of cores, resulting from the action of several glycosyltransferases, can be found in the different oligosaccharides of human mucins (Table 4), even in the mucins secreted by a single individual [ 11 1-1 161.

3 62

Table 4 Human mucin cores

Respiratory Salivary Gastric Colonic Meconium Amniotic fluid Ovarian Cervical

1

2

3

+ + +

+

+ + + +

+

+

+ + + + +

Core types 4

+ + +

+

103 100,101

+

+

102,108,121 109

106,107

-

GaN

Gnp 1

I

G D ~ Core7

1

\

‘GaN

GP 1 core 2

-

Core 6

GaN

J

GaNui 6

GaN

Core 7

’

paNCore 3

Gnpl

Gnp1

GaN-

, \

Core 5

lr +

+-

3GaN-

/

+

99,95

GaNu 1

\6

246

+

GaN

Gnp 1

117,122 105,110

f ,3

/

Ref

8

+ + +

+ +

+

6

5

-

-

‘ 6

,FaNGnpi

I paN

/

Core4

-

Core8 Fig. 5. Human much core structures. GaN, N-acetylgalactosamine; G, galactose; Gn, N-acetylglucosamine.

363

i ag

Fig. 6. Examples of 0-glycan backbones produced by linear or branched elongation. They can correspond to polymers of two types of disaccharide subunits, type 1 [Gal(@1-3)GlcNAc] or type 2 [Gal(pl+GlcNAc]. G, galactose; Gn, N-acetylglucosamine. Glycosidic linkages are represented as follows: /, p- I ,3- linkage; -, p- 1,4- linkage; \, p-1,6-linkage.

2.4.3. Carbohydrate chain elongation Synthesis of the backbones of different carbohydrate chains results from the successive action of glycosyltransferases allowing the transfer of galactose or N-acetylglucosamine into a determined position and anomeric linkage. Mucin carbohydrate backbones are made up of disaccharides formed by alternating galactose and N-acetylglucosamine residues, always P-linked, with two types of linkages: Gal(P1-3)GlcNAc (type 1 disaccharide) or Gal(~1-4)GlcNAc(type 2 disaccharide) [98,117,118,123]. During elongation of the carbohydrate chains, these two disaccharide units can start from each of the cores or be linked P1-3 andor Pl-6 to an internal galactose residue of the backbone to give branched or linear backbone structures [98,117] (Fig. 6). Polymers of the type 2 disaccharide units in GlcNAc(P1-3) linkages generate i antigens. Branched type 2 disaccharide units give I antigen. 2.4.4. The periphery of carbohydrate chains The periphery of the mucin oligosaccharide chains is characterized by the presence

3 64

G-Gn Fa1/ 3 x H’

Leb

G8

Gn

-

G Gn 2

2

H2

Fa;

Fa; F- Gn G/

G -Gn Y

”

1

FF

F

G -Gn

G~N/~F/ G

’

AY

G -Gn

’

FF

By

Fig. 7. Examples of peripheral regions stemming from various substitutions of type 1 or type 2 disaccharide units. G, galactose; Gn, N-acetylglucosamine; GaN, N-acetylgalactosamine; F, fucose. Glycosidic linkages are represented as follows: /, (3-3 linkage; dashed /, al-3 linkage; -, p-1,4- linkage; \, 8-1,6- linkage; dashed I, a-1,2- linkage.

of sugars such as Fuc, Gal, GalNAc, NeuAc, most often in a anomeric configuration. Sulfate can also be added to the periphery of mucin chains. These sugars are added by different glycosyltranferases, genetically controlled and may confer blood group antigenic activities to the mucin (ABH, Secretor, Lewis) [118,124-1291. At least two types of fucosyltransferases are involved in the biosynthesis of mucins, the Secretor enzyme and the Lewis enzyme and the A and B enzymes are responsible for A and B activities [ 1301321. Fucose may be added to terminal type 1 or type 2 disaccharides to generate different structures, H1, Lewis a, Lewis b, H2, X and Y (Fig. 7). Oligosaccharides containing fucose residues linked al-2 to the galactose of a type 2 disaccharide in an internal position in the backbone, have been isolated these fucose residues are responsible for new structures called “internal H ’[ 1161 (Fig. 8). A, B, H and Lewis b determinants are not expressed in mucins from non-secretor individuals [ 122, 1331. In urine, besides the Tamm-Horsfall glycoprotein, a mucin carrying Sda or Cad speci-

365

Leb-H2

type 2 - H2

Fig. 8. Examples of internal fucosylation. The oligosaccharides have an internal H2 structure.

ficity has been isolated from pooled blood group 0 urines [134]. Such an activity has also been found in saliva [ 1351. Numerous carbohydrate chains carry acidic groups, either sialic acid or sulfate, responsible for the polyanionic character of mucins. Different types of structures have been identified where sialic acid is linked either to the N-acetylgalactosamine of the carbohydrate-peptide linkage or to a terminal galactose implying a-2,3-Gal-, a-2,6-Gal-, and a-2,6-GalNAc sialyltransferases [93,118, 1361 (Fig. 9): NeuAc(a2-6)GalNAc(a 1-0)peptide NeuAc(a2-3)Gal(P 1-3)GalNAc(a 1 -0)peptide Gal@ 1-3)peuAc(a2-6)]GalNAc(a 1-0)peptide (sialylated core 1) Gal(al-3)[NeuAc(a2-6)]GalNAc(a 1-0)peptide (sialylated core 8) NeuAc(a2-3)Gal(fi 1-4)GlcNAc-R NeuAc(a2-6)Gal((3 1-4)GlcNAc-R Most human mucins contain only N-acetylneuraminic acid. In the colonic mucosa, sialic acid residues may be 0-acetylated [137-1381. Muchmore et al. [I391 have demonstrated that colonic tissues contain 9-0-acetyl-N-acetylneuraminic acid and that this modification of sialic acid increases shortly after birth. 7,9-di-O-acetyl-N-acetylneuraminic acid has also been detected in these mucins [ 1401. N-glycolylneuraminic acid is not normally found in humans. Sulfated carbohydrate chains have been described in human mucins, showing that sulfate groups are attached to galactose residues, either in 3 [102,141] or in 6 and 4 linkages [97,142], and to N-acetylglucosamine in 6 linkage [142-1451. Sialic acid, sulfate and fucose may coexist on the same carbohydrate chains of human respiratory mucin [I451 (Fig. 10). In mucins from meconium, a terminal N-acetylglucosamine linked a 1-4 to the penultimate galactose has been found in several chains [ 1211.

366

SA a2

\

6

GaN

SA a2 '6

G-Gn-

G-GnSA u2 l 3

Fig. 9. Different types of sialylation on mucin oligosaccharides. G, galactose; Gn, N-acetylglucosamine; GaN, N-acetylgalactosamine; SA, sialic acid. Glycosidic linkages are represented as follows: /, p-I ,3- linkage; dashed /, a-2,3- linkage; -, p-1,4- linkage; \, a-2,6- linkage.

G-Gn,

,GaN

8 F

G-Gn,

G-Gn,

FI

G'

GaN

G -Gn, F' G,GaN

1

F

G-Gn, GNhN

&

I

I

'G-Gn,

:SA I

/Gp,

G-Gn, F' dGaN SA F G/GaN:

yGaN F

SA'

SA'

I

;

SA

SA'

GGn, &GaN

vii

Fig. 10. Possible substitutions of a simple tetrasaccharide (i) from human respiratory mucins. This oligosaccharide may be only sulfated (iii), sialylated (v) or fucosylated (vii). It may also be fucosylated and sulfated (ii), sialylated and sulfated (iv), fucosylated and sialylated (vi), or fucosylated, sialylated and sulfated (viii). F, fucose; G, galactose; Gn, N-acetylglucosamine; SA, sialic acid; S, sulfate. Linkages are represented as follows: /, Gal(PI-3)-, Fuc(al-3)-, NeuAc(a2-3)- linkages or 3-sulfate; -, 0-1,4- linkage; \, p-1,6- linkage; 1, a-1,2- linkage.

367

Finally, it should be stressed that only a small part of the oligosaccharides has been identified so far and that each oligosaccharide from the backbone may be substituted in many ways. For example, 24 derivatives of a simple tetrasaccharide (Fig. 10) have been found in the human respiratory mucins which most probably contain hundreds of different carbohydrate chains. The reasons for the remarkable heterogeneity of carbohydrate chains in several mucins are puzzling. It might result from differences in the glycosyltransferase equipment or sugar nucleotide availability, from one cell to another. There might be differences in glycosyltransferase expression from one mucin-secreting cell to another. This is true for sialylation in the airways: limulin lectin which recognizes some sialylated structures but not others has more affinity for the goblet cells than for the mucous glands [18]. There might be a modification of the expression of glycosyltransferases during cell life. Different apomucins might be glycosylated differently. Podolsky [ 1011 has separated distinct species of human colonic mucins by anion-exchange chromatography and has shown that they contained different mixtures of oligosaccharides. 2.4.5. The non-orthodox presence of N-glycans Low amounts of mannose, a sugar residue typical for N-glycans, are frequently observed and this was considered for a long time to be the result of an incomplete mucin purification. The recent discovery of possible sites of attachment for N-glycans in the amino acid sequence deduced from different apomucin cDNAs [54,65,66,69], suggests that N-glycosylation also occurs in several mucins, and that these mucins contain a few N-glycans in addition to the hundreds of 0-glycans (Fig. 4). Ohara et al. [86] have recently demonstrated the presence of N-glycans in a MUC2 mucin synthesized by HM3 human colon cancer cells. 2.4.6. Biosynthesis and secretion This part will not deal with mucin 0-glycosylation [146] which is reviewed in chapter 5.3, volume 29a of this series. The synthesis, transport and secretion of mucins have been studied with radiolabelled precursors in rectal mucosa: even in individual cells, mucus granules do not move in concert to the apical cell surface [147]. Studies have appeared concerning the biosynthesis of human mucins in bronchial [47, 481, gastric [49], gall-bladder [50] and colonic explants [ 148,1491. The precursors were polydisperse and in the range of 200400kDa for human respiratory mucins, more discrete and around 500 kDa for the gastric and gall-bladder mucins [49,50], and 550 kDa for colonic mucins [ 1491. N-glycans were added onto the gastric and gall-bladder mucin precursors before 0-glycosylation; these mucins also formed disulfide-linked oligomers before being secreted. The biosynthesis of colonic mucins has been studied in different human colon cancer cells [86,150,151]. Using HM3 human colon cancer cells, Ohara et al. observed that two precursors were synthesized corresponding to MUC2 and MUC3 gene products, and that the MUC3 precursor was completely degraded by trypsin, unlike the MUC2 precursor that had a 240 kDa resistant fragment containing N-linked carbohydrate; moreover the soluble intracellular mucins ( M , = 5 x 1O6 kDa), were not sensitive to reduction

368

unlike the secreted mucins ( M , > 10'kDa) [86]. Using LS180 cells, derived as were the HM3 cells, from the same LS174T cell line, McCool et al. [150] observed first a monomeric precursor ( M , > 670 m a ) , already weakly glycosylated, followed by thiolsensitive oligomers, implying that disulfide-dependent oligomerization was an essential prerequisite for secretion. Finding good models to study mucin secreting cells is a serious problem. Culture conditions influence mucin synthesis and secretion [ 1521. Many cell lines originating from adenocarcinoma synthesize more hyaluronic acid or proteoglycans than mucins. The glycosylation machinery of such cells may be altered and, even with normal cells in secondary culture, their phenotype may be different from that of cells in their normal mucosal environment. For instance, mucin-secreting cells obtained from the human respiratory mucosa can be grown in secondary cultures. However these cells, which synthesize mucins like typical goblet cells, also secrete lysozyme and mucous proteinase inhibitor, like serous cells [153]. Therefore they have both a serous and a mucous phenotype and probably correspond to a progenitor cell-type. Finally mucosae express several mucin genes but there is very little information concerning the glycosylation of a specific apomucin. Wesley et al. [ 1541 found differences in core peptides of neutral and acidic species of human intestinal mucins. Large biliary mucins having long fucosylated polylactosamine chains and chains carrying sialyl-Lea epitopes have been recently isolated by Baeckstrom [4]. After deglycosylation, these mucins strongly reacted with a MUC3 antiserum and not with anti-MUC2 or MUC4. However the authors did not check for the presence of peptide epitopes corresponding to other mucin genes such as MUC5B which is strongly expressed in gall bladder [155]. 2.5. Functions of secreted mucins

The main features of mucins are their filamentous nature and their extraordinary diversity at the carbohydrate and peptide levels. Besides the highly hydrophilic T-domains, mucins also have hydrophobic parts. They may be uncharged or polyanionic. They interact with each other and are capable of forming entanglements. They also interact with other secreted mucus components (proteins, peptides, lipids) and, together, they are responsible for the visco-elastic properties of mucus. In the gastrointestinal tract, mucins are able to adhere to particles and may have a role as biological lubricants, facilitating the movements of bacteria, sloughed off cells and non-digested particles [9]. In the stomach, the mucus forms a layer over the surface epithelium and acts as a diffusion barrier. Hydrogen carbonate ions are trapped in this layer and allow the formation of a pH gradient from pH 1-2 in the gastric lumen to pH 6-7 at the surface of the gastric epithelium. Due to the viscosity of this layer and to the pH > 4, HCl secreted by parietal cells can penetrate the mucus layer, forming narrow viscous fingers. In the lumen, at pH 2, the narrow fingers cannot be formed, preventing back diffusion of HC1[ 1561. In the airways, mucus and mucins are essential for the efficiency of the muco-ciliary escalator which traps and allows the removal of inhaled particles, keeping the lower airways sterile.

369

Mucins may interact with other molecules of the mucus: lipids [30,157], proteins such as lysozyme [ 158,1591, or mucus protease inhibitor [159,160] are frequently strongly bound to mucins, although through non-covalent but strong interactions. The result of these interactions is probably very important for the rheological properties of the mucus and also for the protection and life-time of some of these molecules. Mucins contain several carbohydrate chains which, in other glycoproteins, are known to interact with different cells. This is the case for NeuAc(a2-3)Gal(fi1-3)GalNAc, a receptor of the macrophage sialoadhesin [ 16 I , 1621, NeuAc(a2-3)Gal(P 14)GlcNAc, a receptor of CD22b, a sialic acid-specific lectin from a subset of IGM+ B cells [163] and sialylated Lewis x determinant, a receptor for selectins [ 1641. Whether or not these determinants are in the proper environment and conformation is totally unknown, but one may speculate on the interactions between mucus and the cells it may encounter during an inflammatory process. Mucins may also interact with exogenous molecules and microorganisms. The fundamental interactions between mucins and microorganisms will be discussed in section 4. However mucins are probably able to bind to many exogenous molecules. For instance, human neutrophil elastase, a product of the inflammatory response, can be inhibited by human airway mucins [ 1651. In lung infection, aminoglycosides are sometimes delivered as aerosols, and it has been shown that acidic mucins could bind aminoglycosides and, to a certain extent, block their antibiotic properties on bacteria [ 1661.

3. Ep isia1in, an epithelia 1 membrane-associa ted glycoprotein There are membrane-associated “mucins” as opposed to secreted mucins and the best example of these mucin-like glycoprotein is episialin, a component of human milk fat globules (HMFG). Various monoclonal antibodies prepared against HMFG membranes, breast cancer cells and membranes from metastatic cells were found to react with this transmembrane type 1 glycoprotein. Episialin [167] is also known by several other names such as polymorphic epithelial mucin (PEM) [ 1681, and polymorphic urinary mucin (PUM) [ 1691. Episialin has an apparent molecular mass of over 300 kDa and more than 50% of the molecule (by weight) consists of 0-linked carbohydrate chains. cDNAs encoding this glycoprotein were cloned from breast and pancreatic cancer cell lines allowing the determination of its complete amino acid sequence [ 167,168,17&174]. This MUCl apomucin, or episialin, contains a 13 amino acid signal sequence, followed by a large sequence corresponding to the extracellular domain of the protein. This domain is mainly characterized by a sequence of nearly identical repeats of 20 amino acids containing 25% hydroxylated amino acids, which is flanked on both sides by shorter non repeated segments. The extracellular domain, which does not contain cysteine [ 1751, is followed by a transmembrane region and a 69 amino acid intra-cytoplasmic C-terminal region. Unlike secreted mucins, episialin contains a single cysteine residue located in the cytoplasmic region. The MUCl gene has been localized on the q2 Lq24 region of chromosome 1 [ 1761. The variable number of tandem repeats (VNTR), from 30 to 90, causes the polymorphism

370

of this MUCl glycoprotein [ 1691 and two types of transcripts may be generated by alternative splicing [ 172,1771. Several possible transcription regulation sites of the MUCl gene have been identified on the 5' flanking sequence [ 1781. A whole range of monoclonal antibodies reactive with the VNTR of this MUCl apomucin have been prepared, such as the DF-3 [179] and SM-3 [ 1801 monoclonal antibodies [6]. The extracellular domain of episialin has multiple sites of 0-glycosylation and a few sites for N-glycosylation on the non repeated segment which is on the C-terminus side of the VNTR. Few studies have been performed on the carbohydrate part of normal HMFG glycoproteins. In human skim milk mucins, Hanisch et al. [181,182] have found 0-glycans ranging from 2 to 16 sugars, having a type 2 core and a polylactosamine linked to the C6 of the linkage-GalNAc; in the polylactosamine chains, the repeating units are linked either by a GlcNAc(P1-3)Gal or a GlcNAc(B1d)Gal sequence: Galb( 1-3/4)GlcNAcP( 1-6) [Gal@ 14)GlcNAc], (fi 1-6)

\

GalNAc. -Gal(b1-3)

I

These results are at variance with those of Hull et al. [ 1831 who have mostly characterized tetra- or penta-saccharides. However, Burchell et al. [ 1841 have recently developed a cell line (MTSVI-7) from normal human milk cells and observed a high GlcNAc to GalNAc ratio in favour of the data reported by Hanisch and coworkers [181,182]. When it is fully glycosylated this membrane-associated mucin has a rod-like conformation and extends far beyond the plasma membrane. During biosynthesis, the extracellular domain is cleaved by proteases but remains associated with the membrane by non-covalent interactions. This glycoprotein is normally expressed as an apical membrane glycoprotein in the breast and found in milk fat globules. In mammary glands, its level of expression is greatly increased during pregnancy and lactation. It is also expressed in pancreas, kidney, and genital ducts as well as on the surface of several glandular epithelial cells (salivary, gastrointestinal, respiratory) [ 185,1861. Similar to secreted mucins, episialin may have protective functions against aggression from the environment. Unlike secreted mucins, it has anti-adhesive properties and is normally expressed at the apical part of the cells; as it sticks out far from the membrane, its high density at this site may prevent cellular adhesion by masking adhesion molecules [2]. A soluble factor, designated as mucomodulin and present in normal colon conditioned medium, has been found to up-regulate the synthesis of MUCl gene by acting on a mucin responsive element: this element is situated between positions -531 and -520 of the 5' flanking region of the MUCl gene [187].

4. Mucins and microorganisms Mucosae, such as the respiratory, gastrointestinal or uro-genital mucosae, are complex

371

tissue organizations forming a barrier between the “milieu inttrieur” and the outside. Through mucus and mucins, mucosae are permanently exposed to a microbial environment, and exchange with it or react to it. Host-microorganism interactions are complex. They may lead to an almost complete sterility of the mucosal surface, such as in the lower airways, where the mucociliary clearance eliminates the inhaled microorganisms. Conversely, there is a well tolerated and physiological colonization of the digestive and genital tracts by microbes. The intestine harbours a wide variety of bacterial strains. In this case, the microbial flora protects the underlying mucosa against pathological strains. This flora also allows the physiological mucolysis of all the mucins secreted by the respiratory tract, the salivary glands and the gastrointestinal tract. First, airway mucus is mobilized up to the pharyngea by the ciliary beating. There, it is swallowed with the saliva. These secretions mix with the different types of mucus secreted along the gastrointestinal tract, and the mucins are finally degraded in the rectum by different enzymes (glycosidases, sulfatases, 0-acetylesterase) secreted by the local flora [ 188-19 I]. At least two microorganisms are capable of using mucus as their sole source of energy: Ruminococcus torques and Bifidobacterium b$dum [189]. There is no mucin left in the faeces, unless the organism lives in a germ-free environment. Infection corresponds to an abnormal colonization by a pathogenic strain that overcomes the normal defences, including the microbial barrier, and induces primary or secondary (inflammatory) alterations of the underlying mucosa. A number of factors are involved in host-microorganism interactions. In the physiological colonization, microbes may harbour surface adhesins or hemagglutinins to attach onto the mucus and live there. Pathogenic strains responsible for infection also have adhesins or hemagglutinins. They may eventually recognize the host cell surfaces. As already mentioned, they may secrete pathogenic factors, enzymes and toxins. In addition, viruses and certain bacterial strains may invade the mucosal cells by using very sophisticated mechanisms. Faced with such pathogenic strains, the host reacts first by using its immediate molecular defences (secretion of mucins hindering access to cell surfaces, iron-chelating molecules, antibacterial peptides such as lysozyme or defensins, antibodies, . . . ) and then by activating its cellular defences (macrophages, immune, and inflammatory cells). Using different assays (microtiter plates adhesion assays [ 1921 and liquid phase adhesion assays [ 1931, a growing number of microorganisms have been found to bind mucins in uitro [ 194-2 161 (Table 5 ) . The sites recognized by adhesins or hemagglutinins of microorganisms are frequently carbohydrates. They are sometimes expressed on both mucins and host cell surfaces and are possible sites of attachment and colonization for these microorganisms 1212,2 17-2281 (Table 6). A given microorganism may sometimes express several different adhesins. The wide diversity of carbohydrate epitopes encountered in most human mucins may be envisaged as a mosaic of carbohydrate chains that are possible sites of attachment for microbes and therefore protect the underlying mucosae. For instance this carbohydrate diversity allows the trapping of inhaled bacteria by the airway mucus blanket and their removal by the mucociliary clearance: this is an immediate and major defence of the respiratory mucosa.

312 Table 5 Microbes adhering to human mucins Microorganisms

Candida albicans Entamoeba histolytica Helicobacter pylorii Hemophilus infuenzae non-typeable Pseudomonas aeruginosa Pseudomonas cepacia Staphylococcus aureus Streptococcus mutans Streptococcus gordonii Streptococcus oralis Streptococcus rattus Streptococcus sanguis Streptococcus sobrinus Streptococcus salivarius Yersinia enterocolitica lnjuenza virus Rotavirus

Respiratory [ref.]

Salivary [ref.]

Mucin Gastric [ref.]

+ [I941

+ [199-2011 + [192,202,205] + [206] + [207,208]

+ [I971

+ [198]

+ [203]

Intestinal [ref.]

Ocular [ref.]

+[195,196]

+ [206]

+ [204]

+ [209] + [210] + [210,21 I ] + [21 I] + [212]

+[210] + [213]

+ [290]

+ [214,215]

+ [216]

Table 6 Carbohydrate structures existing in human mucins and recognized by microorganisms Microorganism(s)

Structure(s)

Ref.

Entamoeba histolytica

Gal/GalNAc N-acetyllactosamine Gal(PI -3)[Fuc(a 14)]GlcNAc GlcNAc(fi1-3)Gal

217a 218 219 220

Candida albicans Streptococcus pneumoniae

GlcNAc(fil-3)Gal(~l-3)GlcNAc(~l4)Glc Actinomyces naeslundii Streptococcus sanguis S-fimbriated Escherichia coli X-fimbriated Escherichia coli Pseudomonas aeruginosa Mycoplasma pneumoniae Influenza virus Injuenza virus ~~~

a

Review.

GlcNAc(P I -4)Gal(D 1-3)GlcNAc@ 1-4)Glc Gal(fl1-3)GalNAc NeuAc(a2-3)Gal(P 1-3)GalNAc

NeuAc(a2-3)Gal(~I-3)GalNAc NeuAc(a2-3)Gal(~1-4)GlcNAc GlcNAc(Pl 4)Gal(D I-3)GlcNAc(~14)Glc GlcNAc(fiI-3)GaI@ 1-3)GlcNAc(B 14)Glc NeuAc(a2-3)Gal(fiI 4)GlcNAc HS0,-0-Gal NeuAc (a2-6) Gal NeuAc(a2-3)Gal

22 1 212 222 223 224,225 226 227 228 228

373

It is possible that the specific features of mucins secreted by a given mucosa, together with other components of the local environment (glycocalyx, membrane-bound glycoproteins and glycolipids), contribute to the formation of a specific ecological niche, allowing the unique proliferation of certain types of microorganisms. Bacteria producing an a-glycosidase able to degrade blood group B determinants are selectively favoured in B individuals [229]. Relationships between Lewis blood group phenotype and recurrent urinary tract infection have been observed in women [230]. However, variability in antigen expression is observed among individuals with the same blood type and secretor status and this may have a significant role in the susceptibility to infection [23 I]. Whether or not these relationships are due to mucins andor to glycolipids or membrane-bound proteins of the local environment is not well established. Interactions may also occur between the unglycosylated parts of mucins and bacteria. Pseudomonas aeruginosa binds to mucin-type carbohydrate determinants [224,225] and also to peptide determinants [205]. 4. I . Caries susceptibility

Colonization and bacterial clearance of the oral cavity are modulated by several salivary components including mucins. A decrease in the proportion of high molecular mass glycoproteins has been described in the saliva of caries-resistant subjects by Slomiany et al. [232]. Nevertheless, salivary mucins play a role in the protection of the oral cavity [233] and have antimicrobial effects against potential pathogens. As seen in Table 5 , salivary mucins may bind many bacteria of the buccal flora. They may be involved in the aggregation of several strains of oral streptococci [21 I]. Moreover, they block the attachment of streptococci to human buccal epithelial cells [234]. 4.2. Ulcer and Helicobacter pylori There is accumulating evidence showing that the colonization of gastric mucosa with Helicobacter pylori is associated with chronic active antral gastritis and peptic ulceration [235], and also with gastric adenocarcinoma [236]. Helicobacter pylori binds to human gastric mucins and sialic acid is involved in the binding [198]. As a matter of fact, a sialic acid-specific adhesin has been cloned[237]. H. pylori can also bind to human gastric sulfomucins [238]. Therefore it is tempting to assign a protective role to the mucins for the underlying mucosa, although, as suggested for other pathogens, the role played by the binding of H. pylori to gastric mucins as a dissemination factor in the gastric mucous layer, cannot be eliminated. The problem is complex because a given mucosa may secrete different types of mucins. The mucous cells of the gastric surface epithelium, and of the deep gastric glands, have a different carbohydrate content: type 1 and type 2 antigens in the surface epithelium and only type 2 in the deep glands [239]. On tissue sections of human gastric mucosa, Helicobacterpylori attaches to the mucous cells of the surface [240], which express the Le and Se genes [239]; they do not attach to the deep glands. This attachment is mediated by the Leb antigen and inhibited by soluble glycoproteins presenting the Leb antigen, such as secretory immunoglobulin A or neoglycoconjugates [24 I]. The Leb determinants

3 74

substituted by galactose or N-acetylgalactosamine (to generate B-Leb or A-Leb) no longer allows bacterial adherence: this might explain why blood group 0 individuals are at higher risk for developing gastric ulcers than A or B individuals. Nevertheless, the precise role (dissemination or protection) played by the different gastric mucins, secreted by the surface epithelium and by the deep glands, respectively, is still an open question. Gastric mucins appear to be altered in patients with gastritis or peptic ulceration [242]. Moreover H. pylori can degrade mucins and undermine mucosal integrity [243]; it has a glycosulfatase active on gastric mucins [244] and can also block mucin exocytosis in the human cell line CL16E [245]. 4.3. Injlammatoly bowel diseases Increased binding of peanut agglutinin (PNA) has been observed in the mucosa of patients with ulcerative colitis or Crohn’s disease [246]. Mucins from patients with ulcerative colitis have an increased expression of T antigens which are concealed in normal colonic much [247] and their neutral sugar content is decreased. The carbohydrate chain length of the colonic mucins is reduced in active colitis [247] and Crohn disease [248]. Similar but enhanced modifications are also observed in adenocarcinoma colonic mucins. Specific alterations in the colonic mucin profiles have been observed in colonic mucus [249] and mucosa [250] from patients with ulcerative colitis. Cofield et al. [I911 have observed a decrease in sulfation and U-acetylation of sialic acid in colonic mucins from patients with ulcerative colitis. Unlike controls, purified mucins from uninvolved colonic mucosal specimens from patients with ulcerative colitis may express 19.9 or DU-PAN-2 carbohydrate antigens [25 11. Although these changes in carbohydrate expression are likely to be secondary to the disease process, they may have important functional consequences since underglycosylated mucins may be more prone to degradation by bacterial mucindegrading enzymes and explain the thinning of the surface mucus layer observed in ulcerative colitis [252]. This would lower the defences of the colonic mucosa, leading to its increased susceptibility to degradation by faecal enzyme extracts [191]. It might also change the spectrum of bacterial colonization. The levels of glycosulfatase and sialate 0-acetyl esterase are higher in the extracts from patients with ulcerative colitis than in healthy individuals [ 1911. 4.4. Cystic fibrosis

Cystic fibrosis (CF) is the most common severe genetic disease among Caucasians. It affects the exocrine glands and, in its most typical form, the main symptoms are chronic pulmonary disease, pancreatic insufficiency and elevated sweat electrolytes (chloride and sodium). In cystic fibrosis, there is a mucus hypersecretion as in chronic bronchitis. Unlike chronic bronchitis, the CF lung infection is very peculiar and characterized by the predominance of Staphylococcus aureus in early life and, rapidly, of Pseudomonas aeruginosa which is almost impossible to eradicate and is responsible for most of the morbidity and mortality of the disease. In the recent years, the prevalence of CF infection by Pseudomonas cepacia has gradually increased and may be responsible for severe degradation of lung function.

375

Cystic fibrosis is due to mutations of a gene localized on chromosome 7 encoding for CFTR (cystic fibrosis transmembrane regulator) (for recent reviews, see refs. [2532561). CFTR is a chloride channel of low conductance activated by protein kinase A. CFTR is an integral transmembrane glycoprotein, localized in the apical membrane of secretory epithelia and contains 1480 amino acids: it has 12 transmembrane helixes, a regulatory domain that may be phosphorylated and 2 ATP-binding folds. Besides its role as a chloride channel, it is quite probable that CFTR has other unknown functions. More than 550 mutations of the CF gene have been described so far [256]. However, in the American and Northern European populations, one mutation, AF508, is found in about 70% of the CF chromosomes and more than 90% of CF patients have at least one AF508 allele [257]. This AF508 mutation is a deletion of a phenylalanine residue at position 508, in the first nucleotide-binding fold of CFTR. No CFTR is found in the apical membrane of patients with a AF508 genotype [258-2601 and the biosynthesis of AF508 CFTR in CFTR-transfected COS cells has been found to be altered[261]: its N-linked oligosaccharides fail to be processed past their immature high-mannose state, suggesting that this mutant protein is trapped within the endoplasmic reticulum. However, when mammalian cells expressing AF508 CFTR are grown at reduced temperature (24-28°C) [262,263] or when AF508 CFTR is expressed in amphibian [264] or insect cells [265], a functional CI- channel activity is observed, indicating that the problem of the AF508 CFTR is more a mislocation rather than a functional abnormality. It has been reported that soluble cytosolic chaperones (hschsp 76) immunoprecipitate with CFTR [266] and recently Pind et al. [267] have shown that calnexin, a calciumbinding transmembrane protein of the ER, plays a role in the quality control machinery of the ER. As a matter of fact, calnexin binds to immature forms of wild-type CFTR, probably by a 2-step mechanism involving first weak lectin-like interactions with the half processed mono-glucosylated N-linked oligosaccharide (Glcl MangGlcNAc*), and then stronger protein-protein interactions with some protein domain allowing the proper folding of CFTR. Once the wild-type CFTR folds, it is deglucosylated and dissociates from calnexin, allowing its translocation and maturation in the Golgi. Interestingly, only approximately 20% of this precursor is converted to the mature complex-type N-glycanbearing CFTR and the remainder is probably degraded [267]. In contrast, AF508 CFTR remains in complexes with calnexin, no mature form is made and all the protein appears to be degraded [267]. The major problem in understanding the pathophysiology of CF is to relate these abnormalities to lung infection by S. aureus and P aeruginosa, the main pathogens encountered in this disease. Several lines of data support a pathophysiological scenario in which either the glycosylation or trafficking machinery of CF cells is abnormal, leading to abnormal glycoproteins (including mucins) and favouring the persistence of P aeruginosa in the airways of CF patients. The mislocation of mutated CFTR may have consequences for other cell compartments. In CF cells, a defective acidification of the trans-Golgiltrans-GoIgi network, of prelysosomes and of endosomes has been observed, leading to modifications in the sulfation and glycosylation processes [268-2701. Bradbury et al. have also demonstrated that CFTR, which is an integral component of the plasma membrane, was implicated in endocytic recycling [271] and existed in clathrin-coated vesicles [272] where it is associated with

376

the a-adaptin of the adaptor complex AP2 [273]. These processes may also be altered in CF. Alterations in glycosylation or sulfation of various glycoconjugates have also been observed in CF. Increased sulfation of CF respiratory mucins [3 1,91,274,275] and of glycoconjugates secreted by CF respiratory epithelial cells in culture [276,277] have been reported. Lo-Guidice [ 1451 has recently described a series of novel sulfated carbohydrate chains in CF respiratory mucins; however, it is still unknown whether or not some of these mucin chains are specific for CF. Lower sialylation has been described for CF intestinal mucin [278] and for proteins secreted by immortalized CF respiratory cell lines [268]. Increased fucosylation has also been observed in intestinal and salivary mucins [203,278], in mucoprotein from the duodenal fluid of CF patients [279] and in plasma membranes of CF fibroblasts [280,281]. Two recent reports using different approaches have demonstrated an increased affinity of Pseudomonas aeruginosa for mucins in cystic fibrosis [ 193,2031. In contrast to a previous study [282], Devaraj et al. [I931 have shown an increased affinity of Pseudomonas aeruginosa for respiratory mucins in cystic fibrosis. Carnoy et al. [203] have made similar conclusions for CF salivary mucins and have shown that the fucose, sialic acid and sulfate contents of CF salivary mucins increased, suggesting an increased glycosylation of certain mucins in CF. Several adhesins localized on the outer membrane of Pseudomonas aeruginosa are able to bind to the carbohydrate part of respiratory mucins [283] but it is not known if they have an increased affinity for CF mucins or if their expression is increased in the dehydrated mucus lining the airways of CF patients. Although there is no evidence showing that mucin modification in CF is a secondary phenomenon related to inflammation, this possibility is still open. It is not known if the expression of mucin genes is modified in the CF mucosae, due for example to inflammation. However, some differences in the non-glycosylated domains of normal and CF mucins have been observed by Desai et al. [284]. Conversely, modifications in the carbohydrate or sulfate content of CF mucins may be a consequence of some specific abnormality in glycosylation, sulfation, or intracellular trafficking of the CF cells. The differences observed from one mucin type to another might simply reflect differences in the biosynthetic machinery of carbohydrate in the different mucin-secreting cells. Elevated levels of “mucin-associated antigens”, carbohydrate in nature, have been reported in the blood of CF patients, but the pathophysiological significance of these findings remains unknown [285,286].

4.5. Infiuenza viruses The binding of influenza viruses to respiratory cells is mediated by viral hemagglutinins which recognize cell surfaces containing terminal sialic acid residues. The hemagglutinins from different strains may differ in their ability to recognize different sialic acidcontaining receptors [287]. Hemagglutinins from human strains of influenza A viruses preferentially recognize receptors with the terminal Sia(a2-6)Gal sequence, whereas hemagglutinins from avian strains preferentially recognize receptors with the terminal Sia(a2-3)Gal sequence [288,289]. These two types of receptors are distributed differently

377

in the tracheal epithelial cells, the Sia(a2-6)Gal sequence being primarily expressed on ciliated cells and the Sia(a2-3)Gal structure being mainly expressed in the goblet cells [290] as well as in their secreted products, the respiratory mucins [93,136]. As a matter of fact, human bronchial mucins are potent binding-inhibitors for viral strains of the avian type recognizing the Sia(a2-3)Gal sequence, but not for strains of the human type recognizing the Sia(a24)Gal sequence [290]. Therefore, the predominance of the Sia(a2-6)Gal sequence on ciliated cells and of the Sia(a2-3)Gal sequence on human respiratory mucins may combine to select for the receptor specificity of human influenza A virus strains [290]. Type C influenza virus has not been associated with as severe a disease as types A and B. Unlike influenza viruses A and B which recognize sequences containing N-acetylneuraminic acid, type C influenza virus recognizes 9-0acetyl-N-acetylneuraminic acid [291], a sialic acid which has not been described so far in human respiratory mucins. 4.6. Leukocyte adhesion dejciency type I1

Leukocyte adhesion deficiency type TI (LAD 11) is a rare syndrome (2 cases) causing severe mental retardation, short stature, recurrent infections, high neutrophil counts with defective neutrophil motility [292-2941, and Bombay phenotype. Unlike patients with leukocyte adhesion deficiency type I who have a deficiency in CD18 neutrophil integrin, these patients express normal levels of CD18 integrin and their leukocytes have a defect in sialyl-Lewis x synthesis that renders the cells unable to adhere to E-selectin. It has been suggested that these patients had a general defect in fucose metabolism and that they were unable to synthesize GDP-fucose. As a matter of fact, we had the opportunity to examine the fucose content of the saliva of one of these patients: we could not find any protein-bound fucose (unpublished data), as might be expected from a subject with the Bombay phenotype not expressing either the blood group H/Se a2-fucosyltransferase or the Lewis type a3/4-fucosyltransferase, or from a subject unable to synthesize GDPfucose.

5. Mucins and tumors Different types of tumors arising from epithelial tissues, benign or malignant, may lead to the hyperexpression of mucin peptides andor mucin-type carbohydrate epitopes. With regard to mucin gene expression, the current data are difficult to synthesize into a general picture for several reasons. As already mentioned, MUC genes correspond to both secreted mucins (MUC2-MUC7) as well as to one membrane bound glycoprotein (MUCI). In most studies on cancer tissues or cancer cell lines, a limited number of genes have been studied. The data are mostly based on histochemical data using monoclonal antibodies, lectins and in situ hybridization, and they are sometimes difficult to correlate with one another (see Table 3). With regard to carbohydrate determinants detected in cancer tissues, cancer cell lines or blood from cancer patients, the problems are even more complex since they may belong to membrane-bound glycoproteins, to mucins or sometimes to glycolipids.

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Nevertheless, a large number of monoclonal antibodies directed against episialin and diverse carbohydrate epitopes, have been produced. Some of these antibodies, as well as lectins, are useful to characterize carbohydrate profiles of a primary tumor that can be correlated with metastatic potential and prognosis [296]. Many studies have been devoted to cell lines derived from various adenocarcinomas but, even among cell lines derived from a given tissue, there may be variations in the expression of the mucin peptides or carbohydrate determinants or both [297]. However, from a medical point of view, these studies are important for their potential diagnostic or therapeutic consequences and we will try to summarize the current knowledge concerning this fast-moving field. 5. I . Breast carcinoma

The expression of the MUCl gene is increased in breast cancer [173,179,298]. Recently a novel MUCI protein, MUCI/Y devoid of tandem repeats, and non-glycosylated, has been found to be expressed in breast cancer but not in tissue adjacent to the tumor [175]. In breast cancer cells, the localization of episialin is no longer restricted to the apical membrane and moreover episialin can be shed. Antibodies directed against the tandem repeat of episialin, such as SM3, show a marked increase in reactivity in tumors. This is due to differences in glycosylation of episialin, exposing peptide epitopes which are normally covered by carbohydrate [ 168,299,3001. In contrast to the normal breast, tumor tissues and cancer cell lines may express other mucin genes such as MUC2 [301], MUC3, MUC4 and the MUCS genes [302]. Twenty years ago, Springer and coworkers [303] described two novel carcinomaassociated antigens, T and Tn antigens, which were found to correspond to Gal@]3)GalNAc(a 1-0)-Ser/Thr and GalNAc(a I-0)-Ser/Thr, respectively. These unmasked epitopes were found on the plasma membrane of most epithelial cell lines derived from breast, lung, pancreas and colon carcinomas. The Tn and T antigens appeared also to be important in the invasiveness of these carcinomas. The carbohydrate part of the MUCl product has been studied in the BT-20 cell line isolated from breast carcinoma [ 1831. The main oligosaccharides found were: (T antigen)

Gal(6 1-3)GalNAc( 01)

Gal((31-3)[Sia(a2-6)]GalNAc(ol)

andlor

Sia(a2-3)Gal(P 1-3)GalNAc( 01)

(sialyl-T antigen)

Sia(a2-3)Gal(~1-3)[Sia(a2-6)]GalNAc(ol). Hanisch et al. [304] have analyzed the glycoforms of MUCl mucins from mammary carcinoma cells. In addition to a decreased glycosylation indicated by a higher reactivity with Mab SM3, they observed a decrease in carbohydrate chain length and in expression of Lea, Le" or sialyl Lea epitopes, and an increase in Tn and sialyl-Tn epitopes. Burchell et al. [ 1841 have recently compared the b- 1,6-GIcNAc-transferase activity responsible for the synthesis of 0-glycan core 2 in different cell lines. This activity was decreased a 100-fold in two breast cancer cell lines as compared to non-malignant cells,

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providing an explanation for the differences in glycosylation between episialin and its glycoforms observed in these cancer cell lines. It has been suggested that Helix pomatia agglutinin (HPA) may be used to evaluate the prognosis of breast carcinoma: HPA-positive tumors have a worse prognosis than HPAnegative tumors [305]. Various peptide or carbohydrate determinants analogous to underglycosylated episialin have been found in the blood from patients with advanced breast cancer. Several antibodies which recognize MUC1 peptide epitopes have been described: CASA, 15.3, HMFG 1, HMFG2, MAM-6 [6,306]. Other antibodies recognize carbohydrate determinants [306]. These tumor-associated antigens may elicit an immune response. Cytotoxic T-lymphocytes isolated from patients with breast carcinoma recognized the underglycosylated peptide core of episialin and this is inhibited by Mab SM3 [307]. Anti-MUC1 protein antibodies have also been detected in sera of cancer patients, using synthetic peptides corresponding to the tandem repeats as antigens [308]. Humans have preexisting antibodies directed against T and Tn, apparently elicited by the intestinal flora, and these antibodies in the presence of complement have a potential to kill carcinoma cells in oitro. A strong autoimmune response against T/Tn epitopes is induced in breast carcinoma: it can be detected by a delayed-type hypersensitivity reaction and by the measurement of anti-T antibodies [303,309]. Springer et al. [309] have demonstrated the interest of the measurement of this response for the detection of incipient breast carcinomas. More exciting, they have vaccinated patients having advanced breast carcinoma with a T/Tn vaccine and reported very encouraging results concerning the efficacy of this treatment [309]. Other potential vaccines bearing sialyl-Tn epitopes are currently under trial [3 10,3111.

5.2. Pancreatic cancer Most pancreatic adenocarcinomas originate from duct cells, the main source of mucins in pancreas. Pancreatic cancers may express MUCl and show a strong expression of MUC2, MUC4, MUCSB and MUC5AC[84]. Pancreas cancer cell lines express high levels of MUC4 and MUCSAC [3 121. The level of expression of MUC2 in cell lines is controversial [84,3 121. A number of antibodies have been generated against pancreatic cancer cells. Some of these antibodies like DU-PAN-2 [3 131 and Span-1 [3 141 recognize sialylated carbohydrate determinants that occur on the MUC1 glycoprotein (episialin) [3 15-3 171. This is also the case for a carbohydrate epitope, CA 19.9 (sialyl-Lea), which was originally described in colon cancer cells [318] and is expressed in pancreatic cancer. The sialyl-Tn epitope is also expressed by pancreatic cancer cells on an unknown apomucin [297]. Recently, it has been suggested that the DU-PAN-2 antigen which is expressed on episialin might also be expressed on another apomucin [312]. These different carbohydrate determinants, as well as others related to the MUC 1 peptide, are elevated in the blood of patients with pancreatic carcinoma [297].

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5.3. Colon adenocarcinoma Different mucin genes have been shown to be expressed in colon carcinomas. Ogata et al. [81] observed that MUCl expressed the same levels as in normal colon, that MUC2 and MUC3 often showed decreased expression, and that MUC4 was sometimes overexpressed. MUC5AC and MUC5B may also be expressed [77]. At the present time, the data concerning the level of expression of these genes are sometimes controversial. There are also differences in the mucin genes expressed by different cancer cell lines [77]. Many histochemical studies have been carried out on carbohydrate antigens expressed in colon carcinoma [77]. A general assumption is that the detected antigens correspond to the carbohydrate chains of mucins, but this may not be true in all cases. There is accumulating histochemical evidence showing an increased expression of the core structures Tn and sialyl-Tn in colon cancer [3 181. The sialyl-Tn antigen seems to be the most sensitive and specific marker for colonic cancer. It may be a good prognostic marker as well: the prognosis of patients with colonic cancers seems to be worse when the tumor is expressing the sialyl-Tn antigen [3 191. The apparent increased expression of sialyl-Tn antigen may be due to the lack of 0-acetylation in colon cancer [320]. The major carbohydrate chains found in human rectal adenocarcinoma glycoprotein were [321]: Sia(a2-6)GalNAc(ol) (sialyl - Tn antigen) (sialyl - T antigen) Sia(a2-6)[(Gal(fi 1-3)]GalNAc(ol) Sia(a2-6)[GlcNAc(P l-3)]GalNAc(ol) (sialyl-core 3) (sialyl-core 5). Sia(a2-6)[GalNAc(a 1-3)]GalNAc(ol) These data are generally interpreted as underglycosylation. The expression of T antigens in colonic adenocarcinoma has been controversial but Campbell et al. [247] have recently demonstrated an increased expression of the Gal@1-3)GalNAc disaccharide in the mucins of those patients, as well as in the mucins from patients with inflammatory bowel diseases, although to a lesser extent. Several groups have started to study the glycosyltransferases involved in the 0-glycan biosynthesis of patients suffering from colon cancer. Yang et al. [322] found that the enzymatic activities, measured in the tumor tissue as well as in the adjacent and distal normal tissue, were extremely variable and did not always correlate with the expression of T, Tn, sialyl-Tn and LeX antigens in tissues or Duke's stage. However there are some general trends. The polypeptide a-GalNAc-transferase activity synthesizing the Tn-antigen was low in most colon cancers [322], explaining the decreased mucin glycosylation that is generally observed in cancer [247,323,324]. Although the polypeptide a-GalNAc-transferase activity was low, the Tn increase in malignant colonic cells has been related to a decrease in the fi- 1,3-N-acetylglucosaminyltransferase (core 3 enzyme) [322,325]. The level of fi-1,3-galactosyltransferase(core 1 enzyme) activity in cancer tissues may increase [322] consistent with an increased expression of the Gal((3-3)GalNAc disaccharide T antigen in the mucins of these patients [247]. The a-2,6-sialyltransferase synthesizing sialyl-Tn, the sulfotransferase acting on core 1 and the fi-1,6-GlcNAc-transferase responsible for the synthesis of core 2,

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core 4 and I antigen are generally decreased, whereas the a-2,3-sialyltransferase and a fucosyltransferase synthesizing the H determinant are increased in colon cancer tissue [322]. In the proximal colon where the ABH blood group antigens are normally expressed, cancer cells may lose the relevant blood group activity. In the distal colon which does not normally express ABH and Leb antigens, a re-expression of these antigens has been observed in tumors, as well as abnormal expression of A blood group activity in certain patients having 0 or B blood group [3 191. An increased expression of Leb, Lex, Ley, and extended forms of LeXor Ley has also been consistently reported. For instance, Hoff et al. [326] have described a sialyl dimeric LeX antigen in colonic carcinoma. Hanisch et al. [327] have defined a new epitope (monofucosylated polylactosaminoglycans) expressed in mucins of amniotic fluid and colon carcinoma. The sialyl-Lea antigen, known as 19-9, is expressed in foetal colonic mucosa and frequently expressed in colon cancers. A loss of 0-acetylation of sialic acid has been observed in colorectal cancer cells [328]. Several interesting studies have been carried out on glycosylation in colon cancer cell lines. Twelve oligosaccharides have been isolated from the mucins secreted by a cell line (CL.16E) derived from colonic cancer cell line HT29[329]. In contrast to oligosaccharides observed in normal colonic mucins which have a core 3 and sialic acid linked a 2 4 to galactose [ 1011 and also a core 1 [247], these novel oligosaccharides have cores 1, 2 or 4 and sialic acid linked a2-3 to galactose residues with sometimes an additional sulfate group attached to C6 of the same galactose residue. Vavasseur et al. [330] have compared three colonic cell lines, an adenoma cell line, intermediate premalignant and tumorigenic lines, derived from the previous one. These three cell lines were used as a model to study changes in 0-glycosylation during the progression to cancer. The phenotype of the original adenoma cell was closest to the normal colon and, during progression to cancer, four activities appeared to be turned off core 3 B- 1,3-GlcNAc-transferase, core 4 B- 1,6-GlcNAc-transferase, I p- 1,6-GlcNActransferase and a-2,6-sialyltransferase.The sulfotransferase acting on core 1 decreased during progression to cancer and there was no 0-acetyl-sialomucin in those cells. However, Tn, sialyl-Tn and sialyl-Lea determinants were not detected in those cells or in their secretory products. Altogether these data may be difficult to understand. There is a possibility that the different carbohydrate determinants are harboured by different glycoconjugates and that the expression of these glycoconjugates varies from one tumor to the other. Nevertheless, several serological tests are used in the follow-up of patients treated for cancer. The 19-9 antigen (sialyl-Lea) is used for patients treated for colon carcinoma. However, increased levels of 19-9 in serum have also been observed in other pathological situations such as ulcerative colitis, Crohn's disease, cystic fibrosis [286] and obstructive biliary disease [331]. 5.4. Rectosigmoid villous adenoma

Rectosigmoid villous adenoma occurs principally in the rectum and sigmoid and has a high frequency of malignant transformation. A recent report by Buisine et al.

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demonstrates the potential interest of studying the MUC genes in patients suffering from this disease [personal communication]. An overexpression of MUC2 was observed in the adenoma together with an expression of MUC5AC, a mucin gene which is not expressed in controls. This aberrant expression of MUCSAC can be used in the early detection of recurrences after endoscopic laser treatment.

5.5. Gastric carcinoma MUCl and MUC5B appear to be expressed in most gastric carcinomas [77]. Tn and sialyl-Tn antigens are frequently found in such carcinomas and there may be, sometimes, an abnormal expression of T and sialyl-T antigens. These antigens are present both in the cytoplasm and on the cell membrane, whereas the expression of Tn in normallooking mucosae is limited to the cytoplasm [332]. Sialyl-Tn is present in goblet cells of intestinal metaplasia and there is an overexpression of sialyl-T antigen in such gastric intestinal metaplasia [333], in contrast to gastric carcinomas where this expression is not so frequent [332]. The value of Tn and sialyl-Tn as markers of aggressiveness of the tumors is controversial [332,334].

5.6. Ovarian tumors Remarkable and historically important structural studies have been performed by the groups of Morgan and Watkins, and of Kabat on the mucins secreted within mucinous ovarian cysts. They have allowed the elucidation of a number of oligosaccharides with blood group ABH activities [124127,129]. The question of mucin gene expression in these conditions as well as the possibility of abnormal carbohydrate structures are, however, open. Using immunochemistry, Tashiro et al. [85] have recently compared the expression of MUCI, MUC2, Tn and sialyl-Tn in normal ovarian tissues as well as benign ovarian adenomas and adenocarcinomas. None of these epitopes was expressed in normal tissues, except MUC1 in the cell apex of the germinal caelomic epithelium. Tn and sialyl-Tn were expressed in a limited number of mucinous adenomas, but in all of the ovarian adenocarcinomas. MUCl was expressed in many benign serous tumors but less frequently in mucinous benign tumors, whereas it was frequently expressed in adenocarcinomas. The expression of MUC2 in patients with rnucinous tumors increased with malignancy. Finally, it was suggested that the coexpression of MUCI and MUC2 in ovarian mucinous tumors might indicate malignancy [85].

5.7. Airway cysts Congenital bronchogenic cysts are uncommon but represent interesting cases. They are believed to arise in the embryogenesis of the airways. They are lined by a mucus-secreting epithelium and contain mucins. The chemical composition of several of such cyst mucins has been studied [335]: large variations have been observed. In some cysts, mucins were neutral or highly sialylated; in others, they were highly sulfated and the extent of sulfation could be much higher than what was observed for secreted mucins obtained from sputum.

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Moreover, the N-acetylglucosaminel-acetylgalactosamine ratio may be very different from secreted bronchial mucins and this is also true for the amino acid content (there may be more serine than threonine). These observations raise the question of the synthesis of oligoclonal populations of mucins in certain cysts and there is currently no information concerning the mucin genes expressed in goblet cells lining the cysts. Equally, the congenital or secondary obstruction of nasal accessory sinuses may lead to mucoceles characterized as accumulated mucus [336].

6. Conclusions Mucosal mucins secreted by human mucosae represent a very broad family of polydisperse high molecular weight glycoproteins. They are encoded by at least 8 different genes and there is some indication that the message produced by a single gene may be complex. The numerous carbohydrate chains that cover the apomucin may be extremely diverse, adding to the complexity of these molecules. There are still large uncertainties with regard to the number of genes encoding apomucins, as well as to the extent of mucin carbohydrate diversity. Very little is known concerning the relation between a given apomucin molecule and its glycosylation. Consequently, there is still a lot of work to carry out on mucin genes, carbohydrate structure elucidation, correlations between carbohydrate and peptide, biosynthesis as well as secretion and regulation. Due to their wide structural diversity as well to their location at the surface of all mucosae, mucins are involved in multiple interactions with microorganisms, and are very important in the protection of the underlying mucosae. Alterations in mucin carbohydrate and/or peptide are progressively described in infectious or inflammatory mucosal diseases, as well as in cysts, adenoma or adenocarcinoma developed from the different mucosae. Elucidation of their pathophysiological mechanisms might lead to new concepts for the treatment of these alterations, such as using synthetic carbohydrate or peptide decoys in order to trap pathogenic microorganisms in infectious diseases, or to immune therapy in the field of cancer.

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