description) Endocervical mucins are central to fertility/infertility regulation as hormone dependent-changes in their viscoelastic properties control sperm and bacterial access to the upper reproductive tract. The ultimate objective of this research is to understand how steroids regulate mucin biosynthesis in the mammalian endocervix.
The aim of this proposal is to clone and characterize the cDNA for the high molecular weight rabbit cervical mucin which has been isolated, purified and partially deglycosylated. This goal will be achieved by 1) deglycosylating the high molecular weight cervical mucin and obtaining microsequence data; 2) using the microsequence data to prepare gene specific probes/PCR primers as tools to clone and characterize the cDNA for the mucin. The high molecular weight cervical mucin will be maximally deglycosylated with minimal or no protein degradation by a combination of enzymatic and chemical steps as follows: cervical mucin will be incubated with neuraminidase (sialidase) to produce asialo-mucin. The asialo-mucin will be treated sequentially with trifluoromethanesulfonic acid (TFMSA)/anisole to remove peripheral sugars, followed by oxidation and beta-elimination. In conjunction with TFMSA/anisole treatment, the asialo-mucin may be treated with exo- and endo-alpha-N-acetylgalactosaminidases to remove GalNAc and Gal---GalNAc residues. Reaction products will be sent to Harvard Microchemistry where in situ digestion, tryptic peptide separation and microsequence analysis will be performed. Microsequence data will be used in a computer search of GenEMBL and SWISS-PROT data bases to verify that the cervical mucin is new. Microsequence will be used to design gene-specific probes for use with conventional cloning techniques, and primers for adaptor mediated PCR, i.e., Marathon TM 5' and 3' RACE (rapid amplification of cDNA ends), to obtain full-length cDNA sequence. Northern blot analyses and RNase protection assays will be used to determine whether the size and number of mRNAs are altered during cervical differentiation and/or in response to steroid hormones. Future direction will include the use of site-directed mutagenesis to provide templates for the synthesis of new proteins whose structural and functional characteristics can be tested in vitro, and the use of the rabbit cDNAs to clone the human cervical mucin.
| Hendrix, E; Hewetson, A; Mansharamani, M et al. (2001) Oviductin (Muc9) is expressed in rabbit endocervix. Endocrinology 142:2151 |
