Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 f 250 and 63,507 * 200). Mass spectrometry also revealed molecular heterogeneity associated with both the LY and p subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the LY subunit contains 0-30% carbohydrate and the p subunit contains 27-30% carbohydrate.Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the LY subunit ( d 4 N , cy*lN, a I z 3 N ) and three in the p subunit (p@N, p'27N, and @'47N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site p@N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site pI4'N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennaq with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated LY and p subunits, including all Ser-and Thr-containing peptides. No evidence was found for the presence of 0-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 1 1-cis-retinal and/or 1 1-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein.Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 1 5 2 0 % of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state I9F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

The cellular retinaldehyde-binding protein (CRALBP) 1 is thought to play a fundamental role in vitamin A metabolism in the retina and retinal pigment epithelium (RPE). Notably, mutations in the human CRALBP gene can result in autosomal recessive retinitis pigmentosa (1). In vitro CRALBP serves as a substrate carrier protein for enzymes of the mammalian visual cycle, modulating whether 11-cis-retinol (11-cis-Rol) is stored as an ester in the RPE or oxidized by 11-cis-Rol dehydrogenase to 11-cis-retinal (11-cis-Ral) for visual pigment regeneration (2). In the RPE and Mü ller cells of the retina, CRALBP carries endogenous 11-cis-retinoids, the isomers of vitamin A utilized for phototransduction. However, CRALBP is not always associated with a retinoid ligand and more than one physiological role for the protein appears likely (3). The protein is also present in ciliary body, cornea, pineal gland, optic nerve, brain, transiently in iris, but not in the rod and cone photoreceptors. CRALBP is expressed in developing retina and RPE before the tissues contain 11-cis-retinoids or the enzyme responsible for generating 11-cis-retinoids (3). Apparently the protein serves functions unrelated to visual pigment regeneration in brain and tissues not involved in the visual cycle and may bind ligands other than retinoids.CRALBP was first detected in retina about 20 years ago and shown to carry 11-cis-Rol and 11-cis-Ral as endogenous ligands (4,5). Structure function studies have defined ligand stereoselectivity and photosensitivity (6), developed a topological and epitope map (7), established in vitro evidence for a substrate carrier function in RPE (8, 9) and produced human recombi-