Neuronal death in the dorsal root ganglion after sciatic nerve injury does not depend on sortilin.

Injury to the sciatic nerve induces loss of sensory neurons in the affected dorsal root ganglia (DRGs). Previous studies have suggested the involvement of the neurotrophin receptors p75 neurotrophin receptor (p75(NTR)) and sortilin, proposing that sensory neuron subpopulations undergo proneurotrophin-induced apoptosis in a similar manner to what can be observed in the CNS following injury. To further investigate this hypothesis we induced sciatic nerve injury in sortilin-deficient mice, thereby preventing apoptotic signaling of proneurotrophins via the sortilin-p75(NTR) receptor complex. Using an unbiased stereological approach we found that loss of sortilin did not prevent the injury-induced loss of DRG neurons. This result demonstrates that previous findings linking p75(NTR) and proneurotrophins to loss of sensory neurons need to involve sortilin-independent pathways and suggests that proneurotrophins may elicit different functions in the CNS and PNS.

Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs.

The glycoprotein MUC15 (mucin 15) was initially isolated from the bovine milk fat globule membrane. The present work demonstrates the existence of immunologically similar proteins ( approximately 130 kDa) in ovine, caprine, porcine, and buffalo milk samples. Purification and N-terminal amino acid sequencing confirmed the presence of ovine and caprine MUC15 orthologs in milk fat globule membranes. Expression of MUC15 in human milk was demonstrated by immunostaining ( approximately 150 kDa) as well as by mass spectrometry. Screening of a human multiple tissue expression array showed abundant MUC15 gene expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions (epidermal growth factor receptor and Src homology 2 domains) are identified in the intracellular region. Implication of the mucin in signal transduction and the potential physiological function of MUC15 are discussed.

Characterization of carbohydrate structures of bovine MUC15 and distribution of the mucin in bovine milk.

The present work reports the characterization of carbohydrate structures and the distribution of the newly identified mucin MUC15, a highly glycosylated protein associated with the bovine milk fat globule membrane (MFGM). Distribution of MUC15 was investigated in various fractions of bovine milk by densitometric scanning of Western blots. In raw milk, MUC15 was shown to constitute 0.08% (wt) of the protein and approximately 1.5% (wt) of the MFGM-associated proteins. Surprisingly, this study showed that in addition to the fat-containing fractions, such as MFGM and buttermilk, MUC15 was present in nonfat-containing fractions as well, such as skim milk and whey. Compositional and structural studies of the carbohydrates of bovine milk MUC15 showed that the glycans are composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid. The carbohydrate was shown to constitute 65% of the total molecular weight, and the molar ratios of the individual sugars to protein of the O-linked glycans were determined. The glycan structures of MUC15 were further studied by enzymatic deglycosylation experiments using different endo- and exoglycosidases as well as a panel of lectins. The N-linked glycans were shown to contain mainly hybrid-type N-glycans. In addition, the N-glycans were shown to be sialylated and contain terminal poly-lactosamine structures. The O-linked glycans were found to constitute some unsubstituted Core-1 structures and a substantial number of sialylated Core-1 O-linked glycans. By comparing the results of peanut agglutinin lectin binding, enzymatic deglycosylation, and monosaccharide composition analysis, we concluded that bovine MUC15 also contains more complex O-glycans containing high amounts N-acetylglucosamine residues. Furthermore, a small subset of the O-linked glycans is decorated with lactosamine on their terminal ends.

Inhibitory effects of human and bovine milk constituents on rotavirus infections.

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.

Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone.

The highly glycosylated protein MUC1 was purified from bovine milk-fat globule membranes by a procedure involving detergent extraction, ion-exchange chromatography and reverse-phase chromatography. The identity of the purified mucin protein was confirmed by N-terminal sequencing and partial amino acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced amino acid sequence demonstrated structural features characteristic for mucins, including an extracellular tandem repeat region with 11 partially conserved repeats (20 amino acids each), a membrane-proximal SEA module, a transmembrane domain, and a cytoplasmic C-terminal region. Monosaccharide composition determinations suggested significant structural differences between O-linked glycans of MUC1 originating from either bovine or human milk. Interspecies differences of the consensus repeat sequence in MUC1 and the physiological functions are discussed.