Carbohydrate cDNAs

The epitope recognized by the antibody DU-PAN-2 is a mucin. Its molecular mass is between 100 and 500kDa, and it is 80% carbohydrate. cDNA for the core protein has been cloned and sequenced, and the predicted amino add sequence reveals a protein of 126kDa containing 1295 amino acid residues with 42 tandem repeats. DU-PAN-2 antigen is found mainly in the glandular epitheiia of the pancreatic and biliary systems and in the breast and bronchial ducts. A lower level of expression is found in cells of the salivary glands, stomach, colon, and intestine. ... 

The peppermint oil gland secretory cell cDNA library has proven to provide a highly enriched source of candidate genes involved in essential oil biosynthesis. A functional genomics approach has successfully been employed to clone genes involved in the mevalonate-independent pathway of isoprenoid biosynthesis and in the peppermint-specific steps producing (-)-menthol and (-)-menthone. The optimization of LC-MS technology to profile phosphoiylated carbohydrates and... 

Fig. 1. A high-throughput platform of the carbohydrate-based microarrays. A high-precision robot designed to produce cDNA microarrays was utilized to spot carbohydrate antigens onto a chemically modified glass slide. The microspotting capacity of this system is approximately 20,000 spots per chip. The antibody-stained slides were then scanned for fluorescent signals with a Biochip Scanner that was developed for cDNA microarrays. The microarray results were subsequently confirmed by at least one of the conventional alternative assays.

Cholesteryl ester transfer protein (CETP) promotes exchange and transfer of neutral lipids such as cholesteryl ester (CE) and TG between plasma lipoproteins [63-65], The function of CETP is illustrated in Fig. 3. CETP is a very hydrophobic and heat-stable glycoprotein with an apparent molecular weight of 74 kDa as determined by SDS-PAGE analysis [66,67], The cDNA from human liver was cloned and sequenced [68], It encodes for a 476-amino acid protein (53 kDa), suggesting that the apparent higher molecular weight is due to the addition of carbohydrate residues by posttranslational modification. 

Fig. 10. (A) Proteolytic fragments derived from laminin and activities found to be associated with them. (B) A domain model for the B1 chain of mouse laminin deduced from the nucleotide structure of cDNA clones. Domains 1 and II are largely helical and probably form a coiled-coil structure with a similar portion of the B2 chain. There are several possible carbohydrate attachment sites. These domains are separated by a region a" containing six cysteines closely bunched, possibly involved in cross-linking to the B2 and A chains. Domains III and V are cysteine-rich regions composed of repetitive segments of about 50 amino acids each. These domains may form the two rod-like elements within the short arm, whereas domains IV and VI are thought to form the visible globular structures.

LPL is a glycoprotein (8% by weight carbohydrate) with a native molecular weight of around 100 000 Da and a monomer subunit of about 50 000 Da (Kinnunen et al., 1976). Senda et al. (1987) calculated the molecular weight of the unglycosylated form as 50 548 Da based on the cDNA encoding it. LPL has a serine at the active site, located in a beta turn in the enzyme, similar to that at the active site of other serine hydrolases (Reddy et al., 1986). 

The first structure of human renin was obtained from prorenin produced by expression of its cDNA in transfected mammalian cells. Prorenin was cleaved in the laboratory to renin using the protease trypsin. Because the carbohydrates in renin are not required for bioactivity, oligosaccharides were removed enzymatically. This process facilitates crystallization in some cases and also removes the contribution of the heterogeneous sugar chains to the diffraction pattern. The structure was determined without the use of heavy-atom derivatives, by application of molecular replacement techniques based on the atomic coordinates of porcine pepsinogen as the model. The molecular dynamic method of refinement was used extensively to arrive at a 2.5 A resolution structure. However, some of the loop regions were not well resolved in this structure (Sielecki et al, 1989 Sail et al, 1990). 

The presence of carbohydrate chains does not seem to matter for the catalytic activities of the TSAP isozymes, but is of importance for TNAP. The bone and liver TNAP isoforms exhibit the same 507-amino acid sequence but differ in their posttranslational glyco-sylation modifications. The cDNA sequence of ALPL suggests the presence of five N-linked glycosylation sites [2]. In addition, TNAP seems to be O-glycosylated in bone but not in liver. Indeed, the difference between the bone, liver, and kidney isoforms of TNAP is caused by differences in glycosylation, and these posttranslational modifications affect the catalytic activities of these isoforms [37]. 

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