Low molecular mass proteins are implicated in chemical communication throughout mammalian species, being involved in both perception and delivery of pheromonal compounds. In boars, pheromones are secreted in saliva to cause oestrous sows to take up the mating stance. These pheromones are the 16-androstene steroids, 5a-androsten3a-ol and 5a-androsten-3-one. The submaxillary glands of boars contain a low molecular mass protein, pheromaxein, which is capable of binding these 16-androstene pheromones. Pheromaxein was purified, cloned and characterized. It was found to be a nonglycosylated heterodimeric protein, belonging to the secretoglobin superfamily and the major 16-androstene-binding protein present in submaxillary salivary glands of the boar. One subunit, pheromaxein A, was found to be homologous to prostatein peptides, C1 and C2 and lipophilin A and B, whereas the other subunit, pheromaxein C, was homologous to prostatein peptide C3 and lipophilin C. Transcription of pheromaxein A was limited to the prostate and submaxillary salivary glands from both the boar and sow, whereas transcription of the other subunit, pheromaxein C, was more widespread. This is similar to the transcription distribution of lipophilin in humans. Many isoforms of pheromaxein were found to exist, with a molecular mass range of 17 415-18 159 Da; these are probably products of a multigene family. Posttranslational modifications, to generate mature pheromaxein isoforms, probably include C-terminal cleavage of pheromaxein A, followed by additional modifications.Keywords: androstenone; lipocalin; pheromaxein; secretoglobin; steroid-binding protein.Low molecular mass proteins are implicated in chemical communication throughout mammalian species, and are involved in both perception and delivery of pheromonal compounds. The submaxillary glands of the male pig secrete large quantities of saliva when aroused by female pigs. The presence of pheromones within the saliva cause oestrous sows to take up the mating stance. These pheromones are the 16-androstene steroids, 5a-androsten-3a-ol and 5a-androsten-3-one. Biochemical studies have shown that the submaxillary glands of pigs, particularly Go¨ttingen boars, contain a low molecular mass protein, pheromaxein, which is capable of binding 16-androstene steroids [2]. The molecular mass of pheromaxein was estimated as 15 kDa by SDS/PAGE under nonreducing conditions, with two fragments of 8 kDa and 6 kDa under reducing conditions. These fragments were named pheromaxein A and pheromaxein C, respectively, in this study. Two charged isoforms of pheromaxein were identified, with pIs of 4.78 and 5.35, a and b, respectively, each possessing the same molecular mass [3]. When the binding affinities of various steroids to pheromaxein were assessed relative to 5a-androsten-3-one (100%) at 50% binding of 5a-[ 3 H]androsten-3-one, all 16-androstenes tested had strong affinities for pheromaxein, with 4,16-androstadienone the highest at 176%. Other steroids, which had poor binding affinities, were saturated at the C16 ...

CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.

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