ABSTRACT
In an approach toward the identification of hitherto unknown proteins involved in the function of the blood-brain barrier, we constructed a pig brain microvessel-derived cDNA library that is enriched in blood-brain barrier specific sequences by means of subtractive cloning. Sequence analysis of selected clones revealed that one of the cDNAs encoded porcine apolipoprotein (apo) A-1. The identity of apo A-1 mRNA was further confirmed by in vitro translation of RNA from brain microvascular endothelial cells and subsequent immunoprecipitation with an antibody against human apo A-1. We further investigated the expression of apo A-1 mRNA in several tissues and in endothelial cells of the pig. It is shown that cultured brain microvascular endothelial cells provide an in vitro model to study the expression and function of apo A-1 in the microvasculature of the brain.
Subject(s)
Apolipoproteins A/biosynthesis , Cerebrovascular Circulation , Endothelium, Vascular/metabolism , Amino Acid Sequence , Animals , Apolipoprotein A-I , Apolipoproteins A/genetics , Base Sequence , Cloning, Molecular , DNA/analysis , DNA/genetics , Endothelium, Vascular/cytology , Gene Library , Microcirculation , Molecular Sequence Data , Precipitin Tests , Protein Biosynthesis , RNA, Messenger/analysis , Swine , Tissue DistributionABSTRACT
In order to understand the mechanisms regulating the blood-brain barrier we isolated a cDNA clone for gamma-glutamyl transpeptidase from purified porcine brain capillaries by cross-species DNA hybridization using a DNA fragment from a rat kidney cDNA clone. The sequence of the porcine gamma-glutamyl transpeptidase cDNA shows 84% identity in the coding region to the sequence of human placenta gamma-glutamyl transpeptidase cDNA. The derived protein sequences of the human and porcine gamma-glutamyl transpeptidase are 98% similar. Alignment of the porcine and the published rat gamma-glutamyl transpeptidase cDNA sequences revealed an average identity of 79%, except for a small region of 194 base pairs containing a high number of mismatches. After four nucleotide changes within this region of the published rat cDNA sequence, which correct two frame shifts, the derived protein sequences of the rat and the porcine gamma-glutamyl transpeptidase show 97% similarity. The corrected sequence, which includes a region of 65 amino acids, contains additional N-glycosylation sites as well as a hydrophobic protein domain that might represent a second membrane-spanning domain. Tissue-specific expression and diversity of the gamma-glutamyl transpeptidase mRNA transcripts in various porcine and human tissues were analysed by Northern blot hybridization. The 2.2-kb transcript of brain gamma-glutamyl transpeptidase is mainly expressed in the endothelial cells of brain microvessels, in agreement with previous data that showed the brain protein being localized within the blood-brain barrier.
Subject(s)
Cerebrovascular Circulation , Cloning, Molecular , gamma-Glutamyltransferase/genetics , Amino Acid Sequence , Animals , Base Sequence , Capillaries/enzymology , DNA/genetics , Female , Humans , Kidney/enzymology , Molecular Sequence Data , Placenta/enzymology , Pregnancy , RNA/genetics , RNA/isolation & purification , Rats , Sequence Homology, Nucleic Acid , SwineABSTRACT
The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.
Subject(s)
Dendrites/ultrastructure , Microtubule-Associated Proteins/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/embryology , Cell Compartmentation , Hippocampus/physiology , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/ultrastructure , Molecular Sequence Data , Molecular Weight , Nucleic Acid Hybridization , RNA Splicing , RatsABSTRACT
A new route for synthesis of a 32P-labeled Edman reagent 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide (PEPITC) is reported. The carrier-free [32P]-PEPITC (specific radioactivity 3.3 X 10(17) Bq/mol) in theory allows the labeling and detection of a minimum amount of 0.1 femtomol of protein. The advantages of the new reagent, which has previously been used for protein sequencing, are demonstrated by gelelectrophoretical and immunological experiments. Its major role is seen in the quantitative monitoring of protein elution from preparative one- and two-dimensional gels. Proteins thus obtained are, after the usual desalting steps, ready for sequencing.
