A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

A cDNA clone for the histamine HI receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G-protein-coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H, histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H,-receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector ; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H,-histamine-receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine HI receptor mRNAs of 3.5 kb and 4

The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity.z 1998 Federation of European Biochemical Societies.

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