Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression.

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Regional distribution of human trypsinogen 4 in human brain at mRNA and protein level.

Gene PRSS3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. Mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mRNA for trypsinogen 4 has recently been identified in brain and other human tissues. We measured the amount of trypsinogen 4 mRNA and the quantity of the protein as well in 17 selected areas of the human brain. Our data suggest that human trypsinogen 4 is widely but unevenly distributed in the human brain. By immunohistochemistry, here we show that this protease is localized in neurons and glial cells, predominantly in astrocytes. In addition to cellular immunoreactivity, human trypsinogen 4 immunopositive dots were detected in the extracellular matrix, supporting the view that human trypsinogen 4 might be released from the cells under special conditions.