Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Contactus adherens, a special type of plaque-bearing adhering junction containing M-cadherin, in the granule cell layer of the cerebellar glomerulus.

In the glomeruli of the granule cell layer of mammalian cerebellum, neuronal extensions are interconnected by numerous small, nearly isodiametric (diameters up to 0.1 micron), junctions previously classified as puncta adherentia related to the vinculin-containing, actin microfilament-anchoring junctions of the zonula adherens of epithelial and certain other cells. Using immunofluorescence and immunoelectron microscopy, we have found, however, that these junctions are negative for E- and VE-cadherin, for desmosomal cadherins, and also for vinculin, alpha-actinin, and desmoplakin, but they do contain, in addition to the protein plakoglobin common to all forms of adhering junctions, the plaque proteins alpha- and beta-catenin and the transmembrane glycoprotein M-cadherin previously found as a spread--i.e., not junction bound--plasma membrane protein in certain fetal and regenerating muscle cells and in satellite cells of adult skeletal muscle. We conclude that these M-cadherin-containing junctions of the granule cell layer represent a special type of adhering junction, for which we propose the term contactus adherens (from the Latin contactus, for touch, site of bordering upon, also influence), and we discuss the differences between the various adhering junctions on the basis of their molecular constituents.