From carbohydrates to cloning: regulation of gene expression

Abstract

Unusual proteins and glycoproteins rich in proline (PRPs) are products of tissue— specific multigene families. Various biological functions such as calcium binding, hydroxylapatite binding, agglutination of oral bacteria, and formation of acquired dental pellicle have been proposed for PRPs in human saliva. Studies on the glycosylation and conformation of these proteins are presented together with the cloning and regulation of several mouse and hampster PRP genes. Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline, or the so-called proline-rich proteins (PRPs). The PRPs are products of tissue-specific multigene families (1,2) and the cloning and regulation of several mouse and hamster PRP genes will be discussed in detail later. First I will reflect on some other exciting times in my life, especially in regards to glycoprotein chemistry, biosynthesis and immunochemical properties, starting with the synthesis of GDPglucose (3) and UDPGa1NAc (4). I first became interested in glycoproteins as a postdoctoral fellow in Saul Roseman's laboratory. In 1962, Roseman (5) and Warren and Blacklow (6) demonstrated the synthesis of CNP-sialic acid. Subsequently, the sialyltransferase was identified (7,8). By 1963 and 1964, it was shown that sialic acid was transferred from CMP-sialic acid to asialoglycoproteins such as sheep submaxillary mucin (9) and a1-acid glycoprotein (10). These and other studies on glycosyltransferases led to the proposal of multiglycosyltransferase systems by Roseman (11). In 1964, I began studies on a mucoid polysaccharide secreted by Pseudomonas aeruginosa. Doggett and co-workers had isolated a mucoid-type Ps. from the respiratory tract of cystic fibrosis patients (12). This viscous polysaccharide, according to their studies, contained glucose, galactose, glucosamine, galactosamine, sialic acid, and two unidentified substances. The material was negative to the naphthoresorcinol and these investigators concluded that there was g uronic acid present. Subsequent studies from Linker's group (13) and from our laboratory (14) clearly showed that this viscous polymer contained only D-mannuronic acid and L-guluronic acid, the components of alginic acid. Our interests in the chemistry, biosynthesis and immunochemistry of the carbohydrate components of glycoproteins continued with studies on pig submaxillary mucins (15,16) and thyroxine-binding globulin (17,18). About 1965, Elvin Kabat and his co-workers, mainly Gerald Schiffman and Kenneth Lloyd, published a part of their classical studies on the carbohydrate structures of the ABO-blood group substances. Helen Muir, Karl Meyer, Ward Pigman and others had shown that the carbohydrate moieties of proteoglycans and mucins (which include the blood group substances) were attached to serine and threonine via an 0glycosidic linkage. The sugar chains could be removed by an alkali-catalyzed 13elimination (Fig. 1), but with the blood group substances extensive degradation of the oligosaccharides occurred as a result of an additional 13-elimination and of the peeling reaction (19,20). By using the proper alkali and borohydride conditions, essentially quantitative recoveries of the oligosaccharides from pig submaxillary mucins (16) and blood group H substance (21) were obtained for complete structural studies. I of course was very appreciative when Elvin Kabat referred to these conditions as the Carlson reaction (22). The most complex oligosaccharide was the pentasaccharide Oligo-I from PSM, i.e. the mucin from pigs with blood type A. A-PSM was missing the terminal a-linked GalNAc, the primary determinant of blood group A activity. These structural studies were confirmed by the first report on the in vitro synthesis of blood group A antigenic activity (23). The pentasaccharide Oligo-I had both sialic acid and fucose on the same chain, an event unknown before. Proline-rich proteins The primary focus of our research changed in 1975 when we became interested in the synthesis of PRPs. The synthesis of the protein portion plays a dominant and initial role in glycoprotein synthesis. As an overall view of protein synthesis, events from gene to protein (and glycoprotein) are shown in a simplistic manner in Fig. 2. The series of reactions involved in regulating gene transcription and RNA processing are receiving the major attention. Regulation occurs at each major step, however. The first step in cloning usually is the synthesis of complementary copies of the mRNAs by reverse transcriptase or a cDNA library. The cDNAs are cloned and sequenced which gives the amino acid sequence of various proteins. Also, cDNAs labeled with 32P are used to search genomic libraries for specific genes.