Cloning and characterization of human MUC19 gene

Abstract

The most recently discovered gel-forming mucin, MUC19, is expressed in both salivary glands and tracheal submucosal glands. We previously cloned the 3'-end partial sequence (AY236870), and here report the complete sequencing of the entire MUC19 cDNA. One highly variable region (HVR) was discovered in the 5' end of MUC19. A total of 20 different splicing variants were detected in HVR, and 18 variants are able to translate into proteins along with the rest of the MUC19 sequence. The longest variant of MUC19 consists of 182 exons, with a transcript of approximately 25 kb. A central exon of approximately 12 kb contains highly repetitive sequences and has no intron interruption. The deduced MUC19 protein has the bona fide gel-forming mucin structure, VWD-VWD-VWD-"threonine/serine-rich repeats"-VWC-CT. An unusual structural feature of MUC19, which is lacking in other gel-forming mucins, is its long amino terminus upstream of the first VWD domain. The long amino terminus is mostly translated from the sequences in HVR, and contains serine-rich repetitive sequences. To validate the integrity of the MUC19 sequence, primers from both the 3' and 5' end were used to demonstrate a similar tissue expression pattern of MUC19 in trachea and salivary glands. In addition, antibodies were developed against either the amino (N) or carboxy (C) terminus of MUC19, and similar antibody staining patterns were observed in both salivary and tracheal submucosal glands. In conclusion, we have cloned and elucidated the entire MUC19 gene, which will facilitate understanding of the function and regulation of this important, yet understudied, mucin gene in airway diseases.

Figure 1.

Cloning strategy. The top rectangular box represents human MUC19 cDNA: the empty portion is the 5′ end, the patterned potion is central exon, and the filled portion is the 3′ end. Four thick lines directly beneath cDNA represent three major cDNA fragments obtained during the cloning process, and AY236870 is previously reported human MUC19 3′-end partial cDNA sequence. The 5′-end rapid amplification of cDNA end (RACE) products are represented by arrows. RT-PCR products are represented by thin lines flanked by two inward arrows indicating a pair of primers. Three thin lines at the bottom represent three matching EST (expressed sequence tag) clones.

Figure 2.

Characterization of highly variable region (HVR). (A) The top rectangular box represents the partial human MUC19 cDNA; the empty portion is the 5′ end, and the patterned potion is the central exon. Both M19RC6 and M19RC7 are 5′ RACE products. Dashed lines indicate the existence of multiple products. (B) Different HVR transcripts. The 13 rectangular boxes represent different exons. The existence of any of those exons is represented by an “X.” Two altered forms of exon 4 are represented by 4′ and 4″, respectively. And one altered form of exon 6 was represented by 6′. The details of those exons are discussed in the Results section and in Table 3.

Figure 3.

Characterization of transcription start site (TSS). (A) TATA box is marked by underline. An arrow and capitalized letter indicates the TSS. The Kozak sequence is marked by a rectangular box. Nonmatched cDNA: cDNA sequences that don't match with GRCh37/hg19, leading to discovery of the additional genomic sequence, HM801863. (B) The alignment of MUC19 sequences near TSS. c, chimpanzee; h, human; m, mouse; p, pig; r, rat. *Identical nucleotides.

Figure 4.

Analyses of MUC19 protein. (A) The whole rectangular box indicates the entire MUC19 protein of 8,385 amino acids (aa). The dotted box at the very beginning represents long amino terminus (LAT). The three boxes filled with horizontal lines represent three Von Willebrand (VW) D domains. The box filled with diamonds represents the repetitive sequences encoded by the central exon. The box with the grid represents the repetitive sequences encoded by the exons downstream of the central exon. The box with the diagonal lines represents the VWC domain. The filled box represents the cystine knot (CT) domain. The five upward arrows highlight the five classical mucin domains: three VWD domains, one VWC, and one CT domain. (B) The LAT sequence of MUC19 upstream of the first VWD domain. The underlined sequence is putative signal peptide, and the arrow indicates the potential cleavage site. (C) The alignment of the signal peptide among MUC19 and Smgc proteins. The numbers on top (i.e., 10, 20) indicate the position of the amino acid (e.g., 10 represents the 10th amino acid from the left). Periods represent the gap to facilitate the alignment. Similar sequences are marked by gray boxes. h, human; m, mouse; p, pig; r, rat.

Figure 5.

Phylogenetic analysis of gel-forming mucin family. Protein sequences were obtained from GenBank on the following accession numbers: hMUC2, NP_002448; mMuc2, NP_076055; rMuc2, Q62635; hMUC5AC, P98088; mMuc5ac, NP_034974; rMuc5ac, XP_001063331; hMUC5B, NP_002449; mMuc5b, NP_083077; rMuc5b, XP_238988; hMUC6, NP_005952; mMuc6, EDL18119; rMuc6, XP_215127; hMUC19, HM801842; pMUC19, NP_001106757; mMuc19, NP_997126; rMuc19, XP_002729892; Ovomucin, BAB21488; FIMB.1, CAA69604; Spiggin1_1, BAE92619; Spiggin1_2, BAE92620; Spiggin1_3, BAE92621; Spiggin4, BAE92625; hVWF, AAB59458. h, human; m, mouse; p, pig; r, rat. Phylogenetic analysis methods are described in the Materials and Methods.

Figure 6.

RT-PCR analyses of MUC19 expression. The pair of primers for MUC19_3 end is M19RT14, and for MUC19_5 end is M19RT15. Actin was used as a control. All primer sequences are listed in Table 2. Ag, adrenal gland; Bc, brain, cerebellum; Bm, bone marrow; Br, brain (whole); Fb, fetal brain; Fl, fetal liver; He, heart; Ki, kidney; Li, liver; Lu, lung (whole); M, molecular marker; Pl, placenta; Pr, prostate; Sc, spinal cord; Sg, salivary gland; Sm, skeletal muscle; Sp, spleen; Te, testis; Tr, trachea; Ty, thymus; Ut, uterus.

Figure 7.

Representative images from immunofluorescence staining. A total of five fields from four sections (prepared from two healthy individuals) was evaluated. (A–C) are trachea sections, and (D–F) are salivary gland section. (A and D) were stained with the preimmune chicken serum (Pre). (B and E) Stained with hMUC19Ab_N1 (N1). (C and F) Stained with hMUC19_C1 (C1). MUC19-positive images were acquired through fluorescein (green) channel. Propidium iodide (red) was used to stain the nuclei. The white arrow (A–C) indicates the epithelial surface. Scale bar, 100 μm.