*Unité 377 INSERM, Lille, France;
Department of Pharmacology, University of Washington;
Laboratoire de Biochimie et de Biologie Moléculaire de l'Hôpital C. Huriez, CHRU de Lille, Lille, France; and
§Faculté de Médecine, Université de Lille II, Lille, France
Abstract
Mucins, the major component of mucus, contain tandemly repeated sequences that differ from one mucin to another. Considerable advances have been made in recent years in our knowledge of mucin genes. The availability of the complete genomic and cDNA sequences of MUC5B, one of the four human mucin genes clustered on chromosome 11, provides an exemplary model for studying the molecular evolution of large mucins. The emerging picture is one of expansion of mucin genes by gene duplications, followed by internal repeat expansion that strictly preserves frameshift. Computational and phylogenetic analyses have permitted the proposal of an evolutionary history of the four human mucin genes located on chromosome 11 from an ancestor gene common to the human von Willebrand factor gene and the suggestion of a model for the evolution of the repeat coding portion of the MUC5B gene from a hypothetical ancestral minigene. The characterization of MUC5B, a member of the large secreted gel-forming mucin family, offers a new model for the comparative study of the structure-function relationship within this important family.
Introduction
Mucus protects the underlying epithelium from chemical, enzymatic, and mechanical damage. It consists mainly of mucins, which are heterogeneous, highly glycosylated proteins produced from epithelial cells (Ho and Kim 1991
). All mucins contain a central part which carries numerous oligosaccharide chains. This part, rich in Ser, Thr, and Pro, is composed of tandem repeats. The number of repeats and the amino acid (aa) sequence of each repeat depend on the mucin gene. The central part is flanked at both ends by unique domains with aa composition different from that of the repeat domain.
Sequences of the mucin cDNAs are rarely full-length because of the highly repetitive structure and the extremely large size of some mucin messengers. To date, eight human mucin genes, MUC1MUC7 (including MUC5AC and MUC5B) have been well characterized, and each mucosa or secretory epithelium expresses a characteristic pattern of mucin genes. Mucins are usually subdivided into two groups, the secreted mucins (gel-forming and nongel-forming) and the membrane-anchored mucins. The second group consists of the two large mucins MUC3 and MUC4, containing EGF-like motifs, and the small mucin MUC1. MUC6, MUC2, MUC5AC, and MUC5B are the secreted gel-forming mucins, and their four genes are contained within a single 400-kb genomic DNA fragment on chromosome 11 band p15.5 (Pigny et al. 1996a
). At least MUC2, MUC5AC, and MUC5B have a common ancestor (Desseyn et al. 1998a
) and define a subclass of mucins. cDNA sequences flanking the central part of this subclass of human mucins (MUC2, MUC5AC, MUC5B) and animal mucins (RMuc2, FIM-B.1, and PSM) code for cysteine-rich domains which are similar to the cysteine-rich domains that flank the three consecutive A (A1-A2-A3) domains of von Willebrand factor (vWF) (Probst, Gertzen, and Hoffmann 1997
; Eckhardt et al. 1991, 1997
; Gum et al. 1992, 1994
; Xu et al. 1992
; Ohmori et al. 1994
; Lesuffleur et al. 1995
; Desseyn et al. 1997a, 1998b
; Joba and Hoffmann 1997
; Li et al. 1998
; van de Bovenkamp et al. 1998
). These cysteine-rich domains are named D (D1-D2-D'-D3 upstream of the central part in mucins and upstream of the A1-A2-A3 domains in vWF and D4 downstream of the central part in mucins and downstream of the A1-A2-A3 domains in vWF), B, C, and CK (cystine knot; fig. 1
). Partial genomic and cDNA sequences available for the other mucin genes showed that the 3' ends of MUC6 (Toribara et al. 1997
), RMuc5ac (Inatomi et al. 1997
), and BSM (Bhargava et al. 1990
) are similar to the C-terminal regions of the three human mucins MUC2, MUC5AC, and MUC5B.
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A 108-aa subdomain, rich in cysteine residues (10 Cys) and called the "Cys-subdomain," has also been found interrupting several times the central repetitive parts of several mucins. This subdomain has been found seven times in MUC5B (Desseyn et al. 1997a
), twice in MUC2 (Toribara et al. 1991
), at least six times in MUC5AC (Meerzaman et al. 1994
; Guyonnet Dupérat et al. 1995
; Klomp, Van Rens, and Strous 1995
), and several times in various homologous animal mucins (Hansson et al. 1994
; Ohmori et al. 1994
; Shekels et al. 1995
; Turner et al. 1995
; Inatomi et al. 1997
). This Cys-subdomain has been well conserved throughout evolution. The Cys residues and some other amino acid residues are absolutely conserved (Desseyn et al. 1997b, 1998a
), and one putative C-mannosylation consensus sequence (W-x-x-W; Krieg et al. 1998
) is always found in the amino-terminal region of this domain. Because this subdomain is found in humans, mice, and rats, it is likely to play an important function, such as packaging or trafficking, for example, or it may interact with other components of the mucus.
Evolutionary studies of mucin genes can help to define their structure-function relationship and elucidate their individual biological roles. The genomic organizations of the two small mucin genes MUC1 and MUC7 have previously been reported (Lancaster et al. 1990
; Bobek et al. 1996
). We recently published the complete genomic sequence of the large secreted mucin MUC5B (Desseyn et al. 1997a, 1997b, 1998b
). Another group reported a 39-aa-longer amino-terminal region (Offner et al. 1998
) with an additional first exon (which we call 0) and a longer exon corresponding to our exon 1 (which we call 1') for the MUC5B gene. Comparison between the full length (15.8 kb) cDNA sequence and the corresponding genomic sequence (39 kb) revealed a total of 49 exons and 48 introns. The additional intron we call 0, between exon 0 and exon 1', is 2.4 kb long (unpublished results) and is a phase 1 intron. Since MUC5B is the only large mucin gene for which both complete cDNA and genomic sequences have been determined, it provides an excellent model for the investigation of mucin evolution.
Materials and Methods
The accession number of the central part (protein) of MUC5B is CAA70926. Precise boundaries of the different repeats have previously been determined (Desseyn et al. 1997b
). Nucleotide sequences of CK domains are available from the EMBL database with the following accession numbers, and the sequences used for alignment and analysis are defined as follows: human NDP (hNDP): NM_000266, nt 571792; mouse NDP (mNDP): X92394, nt 588809; MUC5AC: AJ001402, nt 29173123; RMuc5ac: U83139, nt 30783284; MUC5B: Y09788, nt 91179970 (join 9172 to 9829); MUC2: M94132, nt 26802877; RMuc2: M81920, nt 22362433; BSM: M36192, nt 15241721; PSM: M61883, nt 32263423; FIM-B.1: J02910, nt 9671164; Human vWF (hvWF): NM_000552, nt 82158418; MUC6: U97698, nt 10331242.
The multiple-sequence alignments were made using the CLUSTAL X program (Thompson, Higgins, and Gibson 1994
) and are displayed by TREEVIEW (Page 1996
).
Results and Discussion
Evolutionary History of the Unusual Large Central Exon of MUC5B
The entire mucin MUC5B gene has been cloned within two overlapping cosmid clones (Desseyn et al. 1997a, 1997b, 1998b
). The mucin-type region (rich in Ser, Thr, and Pro) is composed of irregular tandem repeats of 29 aa (87 bp) in domains called RIRV (Desseyn et al. 1997b
). This mucin-type region is interrupted four times by two associated nontandemly repeated sequences (fig. 1
). This allowed us to design primers to amplify overlapping cDNAs corresponding to the central part of MUC5B. cDNA cloning and sequencing, together with genomic subcloning and sequencing, allowed us to establish that the central part of MUC5B does not contain any intronicunique or tandemly repeatedsequence. We then conclude that the central part of MUC5B is coded by a single unusually large exon of 10,713 bp, and it is then likely that other large mucins have their central parts coded by a single exon. Moreover, this suggests that the central part arose through internal duplications rather than through exon shuffling. The availability of both complete cDNA and genomic sequences of the central part of MUC5B (Desseyn et al. 1997b
) now allows us to trace its evolutionary history. The deduced peptide is composed of three kinds of subdomains, Cys-subdomains (108 aa, 10 Cys), R subdomains (309657 aa, composed of irregular tandem repeats of 29 aa), and R-End subdomains (111 aa). The first three Cys-subdomains (fig. 1
) are followed by four super repeats. Each super repeat is composed of an R subdomain followed by an R-End subdomain and ending with a Cys-subdomain. Each R-subdomain is composed of 11 (the first two and the last one) or 17 tandem repeats of the irregular motif of 29 aa (87 bp) rich in Ser and Thr (fig. 2A
). The four R-End subdomains are rich in Ser and Thr and are very similar to each other, but they do not exhibit any similarity to any other sequence. Another R-subdomain of 23 irregular repeats of 29 aa follows the fourth super repeat. The presence of repeats at different levels suggests that the central part of the gene evolved by successive duplications. Multiple-sequence alignments and phylogenetic trees (figs. 2A and B
) together allow us to propose a model showing how the five tandem repeat blocks RIRV have been made up. The repeat RV-14 has an extra pentapeptide, TTTPT (fig. 2A
), that probably arose through partial duplication of the RV-14 sequence. New multiple-sequence alignments and phylogenetic trees were then constructed without this pentapeptide. This shows that the block made up of the five repeats RV-3RV-8 is highly similar to the block made up of the five repeats RV-18RV-23. Further alignments without either RV-18RV-23 or RV-3RV-8 show that the block RIII-1RIII-6 and the block RV-1RV-6 are similar to blocks made up of the six repeats of other subdomains. Moreover, alignments using RIII blocks or RV blocks and phylogenetic trees (data not shown) show that blocks RIII/V-1RIII/V-6 are more similar to blocks RIII/V-6RIII/V-12 than to other blocks of six repeats. Thus, these analyses and the order of the subdomains that we defined within the central part of MUC5B allow us to propose a diagram showing the evolution of the repeated sequences (fig. 3
). This scheme is in agreement with our previous model showing evolution from a single ancestral gene of the three human mucin genes MUC5B, MUC5AC, and MUC2 (Desseyn et al. 1998a
). A part of an ancestral gene encoding a primordial Cys-subdomain triplicated to give rise to three Cys-subdomains (fig. 3a
). The resulting gene, composed of these three subdomains flanked by unique sequences rich in Cys and found in the vWF gene (see below), duplicated into the two ancestor genes of MUC5AC and MUC5B (Desseyn et al. 1998a
). The primordial repeat of 87 bp of MUC5B duplicated several times to form a block composed of 11 irregular repeats of 87 bp, followed by a unique sequence rich in Ser and Thr coding for 111 aa. The ancestral super repeat (Cys/R/R-End subdomains) duplicated into two super repeats (fig. 3b
). Then, the first six repeats of the second block of 11 repeats duplicated (fig. 3c
). This event was followed by a further duplication en bloc of a region composed of the third Cys-subdomain, the block of 11 repeats, the R-End subdomain, the last Cys-subdomain, and the block of 17 repeats of 29 aa (fig. 3d
). Finally, the block composed of the first 11 repeats of 87 bp, the following R-End subdomain, and the Cys-subdomain duplicated en bloc (fig. 3e
). The five repeats RV-3RV-7 duplicated into the two blocks RV-3RV-7 and RV-18RV-23 (fig. 3f
). A sequence encoding the pentapeptide TTTPT of RV-14 duplicated (fig. 3g
).
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After mucin gene multiplication, further recent duplications within their central part, as suggested above for MUC5B, led to the present genomic organization of the four human mucins. Of special interest is the observation that the tandemly repeated coding sequences of mucins which contain most of the potential O-glycosylation sites are not conserved within species and between species. The fact that each mucin has its tandem repeats more or less conserved strongly suggests that (1) the central part has evolved with a selective pressure to keep Ser and Thr codons corresponding to the O-glycosylation sites and (2) each tandem repeat portion has arisen through internal successive duplications.
The most recent major event is probably the formation of the central repetitive region of each mucin gene, since the repeated sequences are not conserved among mucin genes. The single large exon of mucin genes is highly variable in size and sequence between species and between members of species. Tandem repeats expanded through replication slippage, unequal sister chromatid exchanges, and gene conversion (Vinall et al. 1998
). This does not allow any frameshift changes but allows variability among individuals in the number of repeats, although peptides between two consecutive Cys-subdomains are always about 400 aa long (Desseyn 1997
; Wickstrom et al. 1998
), which correlates well with previous electronic microscopy studies showing the heterogeneity of mucin glycoproteins (Sheehan et al. 1991
).
Genomic Organization of the MUC5B, MUC5AC, and vWF Genes
Comparison of the genomic DNA sequence of MUC5B with its cDNA sequence allowed us for the first time to determine the genomic organization of a large mucin gene (Desseyn et al. 1997a, 1997b, 1998b
; Offner et al. 1998
). These studies revealed a total of 48 introns, 30 introns upstream and 18 introns downstream of the large central exon (exon 30). The work on the MUC5B gene shows that 23 out of the 51 introns of the vWF gene have the same position and class in the MUC5B gene (table 1
), and 9 other introns of MUC5B may be conserved with introns of the vWF gene. Few introns found in MUC5B are not found in vWF, and vice versa. Genomic organization of other human and animal mucin genes (MUC2, MUC5AC, RMuc2, Muc5ac, and FIM-B.1) may be helpful in determining which introns have been gained and which introns have been lost during evolution. Determination of the genomic organization of MUC5B facilitated the determination of the genomic organization of the 3' end of the MUC5AC gene (Buisine et al. 1998a
), which showed that all of the introns found in MUC5AC are conserved (table 1
) compared with MUC5B (position and class). However, unique tandemly repeated sequences identified in some introns of MUC5B have not been found in MUC5AC. This is probably due to insignificant selective pressure on the intronic sequences. In contrast to exonic regions, intron sizes are not conserved between the two genes. Nevertheless, sequences surrounding splice junctions are more or less perfectly conserved between MUC5B and MUC5AC genes and the first 7 bp of some introns are identical, for example. This probably reflects the fact that intronic splice junctions may not accumulate mutations at the same rate as the rest of the intronic sequences. Although almost no data are available concerning the exon-intron organization of other gel-forming mucin genes, we can anticipate that they all probably depict the same overall genomic organization.
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Conclusions
The four mucins clustered to human chromosome 11 have a CK domain and thus form a mucin subfamily. This subfamily can be divided into two subfamilies depending on the presence or absence of Cys-subdomains interrupting the large O-glycosylated domain. The number of Cys-subdomains is characteristic of each mucin, and studies on this domain will help to elucidate physiological functions of mucins.
Further identification of novel mucin genes, cloning of new mucin cDNAs and determination of mucin gene structure will help to characterize the structure-function relationship of mucins. Conserved domains in mucin peptide are most likely to have functional significance, but unique polypeptides should be considered to have been formed during evolution due to differing biological constraints.
Acknowledgements
This work was supported by le Comit;aae du Nord de la Ligue Nationale contre le Cancer and l'Association de Recherche contre le Cancer. J.-L.D. was supported by a fellowship from the Minist;agere de l'Education Sup;aaerieure et de la Recherche.
Footnotes
Claudia Kappen, Reviewing Editor
1 Abbreviations: aa, amino acid(s); bp, base pair(s); BSM, bovine submaxillary mucin; CK, cystine knot; FIM, frog integumentary mucin; kb, kilobase(s); NDP, Norrie disease protein; nt, nucleotide(s); PSM, porcine submaxillary mucin; vWF, von Willebrand factor.
2 Keywords: gel-forming mucin
11p15
tandem repeat
evolution
cystine knot
3 Address for correspondence and reprints: Jean-Luc Desseyn, Department of Pharmacology, P.O. Box 357750, University of Washington, Seattle, Washington 98195-7750. E-mail: desseyn{at}lille.inserm.fr
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