Expression and characterization of disulfide bond use of oligomerized A2-Pancortins: extracellular matrix constituents in the developing brain

Neuroscience. 2005;133(4):947-57. doi: 10.1016/j.neuroscience.2005.03.009.

Abstract

The region-specific characteristics of the extracellular matrix are crucial for diverse functions in the brain. Pancortins/neuron-specific olfactomedin-related glycoproteins are components of the extracellular matrix. They comprise four alternatively spliced variants, Pancortin-1 to -4, which share a common portion, the B part, in the middle of their structure, have two pairs of alternatively spliced 5' regions, A1 and A2, and 3' regions, C1 and C2. Here we demonstrate that in mice, Pancortin-3 (A2-B-C1) and Pancortin-4 (A2-B-C2), which we have grouped together the A2-Pancortins, were transcribed early during the development of the brain in a region specific manner and were expressed very stably in vivo. They are N-glycosylated and secreted. Furthermore, we examined their ontogenetical expression profiles in the developing thalamus using antiserum against the common B region, since transient expressions of their mRNAs were notable there. In the developing thalami, they lasted long in oligomerized form even after the transcription of their mRNAs decreased to an undetectable level. Further analyses revealed that cysteine residues that are located in the common B part are important for homo- and hetero-oligomer formation of A2-Pancortins. When we substituted cysteine residues 45 and 47 with serine residues in that common B part, oligomerization of the A2-Pancortins was highly disturbed.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Age Factors
  • Animals
  • Animals, Newborn
  • Blotting, Western / methods
  • Brain / embryology
  • Brain / growth & development
  • Brain / metabolism*
  • Cell Line
  • Embryo, Mammalian
  • Extracellular Matrix Proteins / chemistry*
  • Extracellular Matrix Proteins / classification
  • Extracellular Matrix Proteins / genetics
  • Extracellular Matrix Proteins / metabolism*
  • Gene Expression Regulation, Developmental / physiology*
  • Glycoproteins / chemistry*
  • Glycoproteins / classification
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunoprecipitation / methods
  • In Situ Hybridization / methods
  • Mice
  • Mice, Inbred ICR
  • Molecular Sequence Data
  • Mutagenesis / physiology
  • Peptide Fragments / metabolism
  • Protein Structure, Tertiary / physiology
  • RNA, Messenger / biosynthesis
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Transfection / methods

Substances

  • Extracellular Matrix Proteins
  • Glycoproteins
  • Peptide Fragments
  • RNA, Messenger
  • olfactomedin
  • Green Fluorescent Proteins