Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus.

A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive sequence has recently been cloned (Shankar, V., M. S. Gilmore, R. C. Elkins, and G. P. Sachdev. 1994. Biochem. J. 300:295-298). In this article, we report additional new sequence derived by 3'-rapid amplification of cDNA ends technique. The sequence corresponds to a stop codon, 3'-untranslated region of 458 bp, a polyadenylation signal, and poly A+ tail, and represents the extreme carboxy terminus of MUC8. A plasmid construct (pAM3) in pBluescript was generated by in-frame ligation of pAM1 to the 479-bp 3'UTR of MUC8. A 5'-end 325-bp fragment of this cDNA subcloned into the protein fusion and expression vector pET28b(+) was used to generate fusion protein under the control of T7 promoter. The purified fusion protein as well as synthetic peptide corresponding to the MUC8 repeat sequence (TSCPRPLQEGTPGS) were used to raise polyclonal antibodies in rabbits. The antiserum to the fusion protein and to the synthetic peptide reacted with the deglycosylated major tracheobronchial mucin. Immunohistochemical studies using the above antibodies localized the MUC8 protein product to submucosal glands in human tracheal epithelium. Furthermore, the gene from which this cDNA is derived, was mapped to chromosome 12 using DNA from a panel of human-mouse somatic cell hybrids. Fluorescence in situ hybridization was used to assign the regional localization to 12q24.3. Since the eight known human mucin genes map to other chromosomes, we have named this gene MUC8, in accordance with mucin gene nomenclature.

Antigenic cross-reactivity of human tracheal mucin with human sperm and trophoblasts correlates with the expression of mucin 8 gene messenger ribonucleic acid in reproductive tract tissues.

OBJECTIVE: To test whether autoimmunity to sperm in men with cystic fibrosis (CF) is a result of cross-reactivity between sperm and carbohydrate sequences of the abnormal CF mucins, we investigated the possible epitope sharing between sperm surface antigens and CF mucin antigens using specific monoclonal antibodies (mAbs) directed to purified CF tracheobronchial mucin-1 (HTM-1) and the expression of tracheal mucin 8 gene (MUC8) mRNA in normal male and female reproductive tract tissues by Northern blot analysis. DESIGN: A panel of mAbs directed to HTM-1 subspecies (types I to V) and polyclonal antibodies (pAb) to native and deglycosylated HTM-1 were tested for their ability to agglutinate motile sperm. An indirect immunofluorescence assay was used to detect expression of cross-reactive HTM-1 epitopes on sperm, term placenta (n = 3), and purified trophoblasts (n = 9). Northern blot analysis was used to detect MUC8 messenger RNA (mRNA) in male and female reproductive tract tissues. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. MAIN OUTCOME MEASURES: The demonstration of cross-reactive mucin at the protein and mRNA levels in reproductive tract tissues. RESULTS: Of the five mucin subspecies, type II, IV, and V mucin-specific mAbs (21.3, 33.3, and 54.1) induced head-to-head agglutination of motile sperm; pAb to deglycosylated mucin had no effect. Sperm agglutination mediated by type IV mucin mAb 33.3 was abrogated completely by D-mannose. Within the term placental villi, type II mucin, was localized to fetal endothelium, type IV mucin was localized to syncytiotrophoblast, and type V mucin was localized to cytotrophoblasts. Immunologic studies correlated with the results of Northern blot analysis, which revealed strong MUC8 mRNA expression in the human testis, placenta, endometrium, and cervix and weak or undetectable levels in the human epididymis, seminal vesicle, ovary, fallopian tube, and uterus. CONCLUSIONS: Both male and female reproductive tract tissues synthesize tracheal MUC8 mucin. Monoclonal antibodies specific to human tracheal mucin subtypes induced "immune-type" agglutination of motile sperm. Therefore, expression of cross-reactive MUC8 mucin epitopes in reproductive tract tissues may contribute to the development of low affinity, carbohydrate-specific, agglutinating antisperm antibodies in the genital tract.