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Isolation and characterization of MUC15, a novel cell membrane-associated mucin
Lone T Pallesen1, Lars Berglund, Lone K Rasmussen
1Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Abstract:
The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length cDNA encoding a protein of 330 amino-acid residues. N-terminal amino-acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids. Database searches showed no homology to any other proteins. A survey of the human genome indicated the presence of a corresponding gene on chromosome band 11p14.3. Isolation and sequencing of the complete cDNA sequence of the human homologue proved the existence of the gene product (334 amino-acid residues). This novel mucin-like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino-acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane-bound mucins, such as a serine, threonine, and proline-rich extracellular region with several potential glycosylation sites, a putative transmembrane domain, and a short cytoplasmic C-terminal. We have shown the presence of O-glycosylations, identified N-glycosylations at 11 of 15 potential sites in bovine MUC15, and a splice variant encoding a short secreted mucin. Finally, analysis of human and bovine cDNA panels and libraries showed MUC15 gene expression in adult human spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, bovine lymph nodes and lungs of both species.
Insights
Researchers identified a novel mucin-like protein, MUC15, in bovine milk and human tissues. This highly glycosylated protein, MUC15, exhibits structural similarities to other membrane-bound mucins and is expressed across various species and tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- The milk fat globule membrane is a complex structure containing various proteins.
- Characterization of novel proteins can reveal new biological functions and pathways.
Purpose of the Study:
- To isolate and characterize a novel protein from bovine milk fat globule membranes.
- To identify and analyze the human homologue of this protein.
- To determine the structure, function, and expression pattern of the novel protein, named MUC15.
Main Methods:
- Protein isolation and purification from bovine milk fat globule membranes.
- Partial amino-acid sequencing to generate oligonucleotide primers.
- Full-length cDNA isolation and sequencing for both bovine and human homologues.
- Database homology searches and genomic localization.
- Analysis of gene expression in various human and bovine tissues.
Main Results:
- Isolation and characterization of a highly glycosylated protein, PAS III, from bovine milk fat globule membranes.
- Identification of a full-length cDNA encoding a 330-amino acid protein, confirmed as mature MUC15.
- No homology found with known proteins, indicating novelty.
- Human homologue identified on chromosome 11p14.3, with a 334-amino acid product.
- MUC15 exhibits structural features of membrane-bound mucins, including a glycosylated extracellular region, transmembrane domain, and short cytoplasmic tail.
- O- and N-glycosylations were confirmed in bovine MUC15.
- A splice variant encoding a secreted mucin was identified.
- MUC15 gene expression detected in diverse human and bovine tissues.
Conclusions:
- A novel mucin-like protein, MUC15, has been identified and characterized in both bovine and human species.
- MUC15 possesses structural characteristics typical of membrane-bound mucins and undergoes significant glycosylation.
- The widespread tissue expression of MUC15 suggests diverse physiological roles in both species.