Effect of Glycosylation on the in Vivo Circulating Half-Life of Ribonuclease

The circulating half-lives of the four isozymes of bovine pancreatic ribonuclease (RNases A, B, C, and D) have been determined in normal and in nephrectomized rats. The isozymes differ only in their glycosyl content. While A contains no sugars, B has a simple oligosaccharide (GlcNAc, Man,+), and C and D each have a complex oligosaccharide (GlcNAc, Man,., Gal, Fuc NeuAc%, and GlcNAc, Mans Gal, Fuc NeuAc,, respectively) attached to Asn-34 of the polypeptide chain. All four isozymes were cleared rapidly in normal rats (t,,, = 2 to 3 min), as expected on the basis of the established role of the kidneys in removing low molecular weight proteins from circulation. In nephrectomized rats, however, a much slower clearance was observed, thus permitting the evaluation of the role of the carbohydrate chains in the catabolism of the isozymes. The clearance curves can be analyzed in terms of two processes, a rapid initial one, shown to represent the equilibration of the injected enzyme into extravascular space, and a second one which is interpreted as the catabolic clearance of the enzyme. The half-life of the RNase isozymes was calculated from this second process and found to be in the range 528 to 577 min for RNase A, 15 min for RNase B, 681 to 862 min for RNase C, and 839 to 941 min for RNase D. The rapidly cleared RNase B was treated with cu-mannosidase to remove 3 of the 4 mannosyl residues, leaving only a trisaccharide (GlcNAc,-PMan) attached to the protein. The half-life of this RNase B derivative was found to be in the range 616 to 733 min. From these results it is concluded (a) that the addition of complex oligosaccharides to a protein does not have any significant direct effect on its circulating half-life (RNases C and D compared to RNase A), and (b) that in the rat there exists a mechanism for clearing glycoproteins based on specific recognition of exposed oc-mannosyl residues (RNase B compared to the other isozymes and to cY-mannosidase-treated RNase B)

Nonenzymatically Glucosylated Albumin: In Vitro Preparation and Isolation from Normal Human Serum

Incubation of human serum with D-[6-3H]glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material. Upon chromatography on Sephadex G-200, radioactivity was found associated with each of the major molecular weight classes of serum protein. Purified human serum albumin was also glucosylated in vitro upon exposure to D-[6-3H]glucose in phosphate-buffered saline. The glucosylated and unmodified albumins were separated by ion exchange chromatography. The physiological significance of these observations in vitro was confirmed by the isolation and quantitation of glucosylated albumin from normal human serum. Glucosylated albumin represents approximately 6 to 15% of total serum albumin in normal adults. The post-translational modification appears to occur by a nonenzymatic process analogous to that responsible for glucosylation of hemoglobin A to hemoglobin AIc, i.e. through Schiff base formation and Amadori rearrangement to a ketoamine derivative

Inulin-\u3csup\u3e125\u3c/sup\u3eI-Tyramine, an Improved Residualizing Label for Studies on Sites of Catabolism of Circulating Proteins

Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells. Overall, these experiments indicate that 125I-InTn should provide greater sensitivity and more accurate quantitative information on the sites of catabolism of long-lived circulating proteins in vivo

The Role of Mannosyl-phosphoryl-dihydropolyisoprenol in the Synthesis of Mammalian Glycoproteins

A mouse myeloma tumor was used as a model system to study the biochemical steps involved in the incorporation of mannose into glycoproteins. This tumor, MOPC-46B, synthesizes a K-type immunoglobulin light chain (K-46) which is a glycoprotein with a single oligosaccharide side chain containing mannose as one of its constituent sugars. MOPC-46B microsomal preparations contain enzymes which transfer mannose from the sugar nucleotide, GDPmannose, to endogenous lipid and protein acceptors. Formation of the mannolipid proceeds by the reversible transfer of mannose from GDP-mannose to an endogenous phospholipid. The mannolipid was purified and characterized by chemical methods and mass spectrometry as a mannosyl-monophosphoryl- dihydropolyisoprenol, containing at least 18 isoprene units, one of which is saturated. The mannolipid was implicated as an intermediate in the in vitro mannosylation of endogenous protein acceptors by three kinds of experiments. (a) Incorporation of [l%]mannose into protein was observed after the initial substrate, GDP-mannose, had been destroyed by sugar nucleotide hydrolases associated with the microsomal preparations. The continued increase in radioactivity in the protein fraction occurred concomitantly with a loss of radioactivity from the mannolipid fraction. (b) I ncorporation of [14C]mannose into both lipid and protein was inhibited by EDTA added at zero time. However, addition of EDTA after mannolipid synthesis had occurred resulted in cessation of mannolipid formation but continued incorporation of mannose into protein to an extent proportional to the amount of mannolipid originally formed. The increase in radioactivity in protein was again accompanied by a loss of radioactivity from the mannolipid. (c) When microsomes were pulsed briefly with GDP-[14C]mannose, which was then chased by a large excess of unlabeled GDP-mannose, incorporation of [‘XZ]mannose into lipid ceased immediately with the chase, while incorporation into protein continued afterwards to an extent proportional to the amount of mannolipid formed prior to the chase. Evidence that the mannolipid could function as a donor of mannose residues to protein was obtained by demonstrating that microsomes catalyze the transfer of [*4C]mannose from exogenously supplied mannolipid to endogenous protein acceptors. The amount of mannose transferred to protein was proportional to both microsomal protein and lipid concentrations. In addition, the amount of mannose transferred to protein from exogenous mannolipid is comparable to that incorporated from an equivalent amount of mannolipid generated endogenously from GDP-mannose. Gel filtration profiles of the [14C]mannose-containing protein formed in this system are essentially identical regardless of whether GDP-mannose or mannolipid is used as substrate. In both cases the radioactive protein fractionates in a manner similar to authentic K-46 (mol wt 24,000). The mannose-containing protein formed from either GDPmannose or mannolipid was degraded sequentially by Pronase and subtilisin. The products formed from either substrate appeared to be identical and exhibited chromatographic and electrophoretic characteristics of glycopeptides. It was concluded that mammalian microsomal preparations contain an endogenous phospholipid, characterized as a dihydropolyisoprenol-monophosphate, which serves as an acceptor of mannose from GDP-mannose, resulting in the formation of mannosyl-monophosphoryl-dihydropolyisoprenol, and that this mannolipid serves as a glycosyl donor for transfer of mannose residues to endogenous protein acceptors. The evidence indicates that the mannolipid is an essential intermediate in the in vitro transfer of mannose from GDP-mannose to protein

Glycation of Amino Groups in Protein

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 “C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na- BH&N and the a-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active sitaec counted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included Ne-Lys-1 (15%), N-Lys-1 (9%), and Lys-37 (9%w) hich are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase

Identification of N epsilon-Carboxymethyllysine as a Degradation Product of Fructoselysine in Glycated Protein

The chemistry of Maillard or browning reactionosf glycated proteins was studied using the model compound, Nu-formyl-W-fructoselysine(f FL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of lVcarboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified a s , the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body

Nonenzymatic Glucosylation of Rat Albumin: Studies in Vitro and in Vivo

Incubation of rat serum with D-glucose in vitro resulted in nonenzymatic glucosylation of serum proteins. Analysis of freshly isolated rat albumin by ion exchange chromatography indicated that the glucosylated albumin accounts for 6.7.+-. 0.9% of total albumin in normal rat serum. Glucosylation of rat albumin in vitro was 1st order with respect to glucose and albumin concentrations and occurs primarily (\u3e 90%) at intrachain lysine residues. Kinetic analysis and inhibition of glucosylation by aspirin suggest that 1 reactive lysine residue is the primary site of glucosylation. Less than 5% of the radioactivity from glucosyl-albumin was released as glucose or mannose by hydrolysis conditions normally used for the analysis of neutral sugars in glycoproteins. Studies in vivo demonstrated that the half-life of albumin in normal rats was unaffected by the addition of 1 mol of glucose/mol of albumin. In addition, glucosylation was a stable modification since 125i-albumin isolated up to 3 days after injection of glucosylated 125i-albumin was recovered only in the glucosylated fraction. In contrast, following injection of unglucosylated 125i-albumin there was a gradual shift of 125i radioactivity to the glucosylated albumin fraction, as would be predicted for nonenzymatic glucosylation occurring in the circulation. Finally, levels of glucosylated albumin isolated from diabetic rats (alloxan induced) were significantly (4-fold) elevated 4 days after withdrawal from insulin therapy. The rat should be a suitable animal model for in vivo studies on nonenzymatic glucosylation of albumin and other serum proteins in normal and diabetic metabolic states

[\u3csup\u3e3\u3c/sup\u3eH]‑Raffinose, a Novel Radioactive Label for Determining Organ Sites of Catabolism of Proteins in the Circulation

The primary tissue sites of catabolism of plasma proteins with long circulating half-lives are unknown. It has been difficult to identify these sites because plasma proteins are delivered to tissues at relatively slow rates but are rapidly degraded intracellularly within lysosomes. Therefore, a tracer attached to protein is lost from the site of uptake before an amount sufficient for quantitation can accumulate. We hypothesized that sucrose (Glupal-2 /3Fruf) would be a useful label to circumvent this difficulty because of the stability of sucrose in lysosomes; and thus, sucrose should remain in tissue long after the protein to which it was attached had been degraded to products released from the lysosome. [G-3HJRaffinose (RAF, Galpal- Glupcwl- 2flFruf) was selected as the vehicle for attaching sucrose to protein. [31XjRAF was converted to the C-6 aldehydogalactose form with galactose oxidase and then covalently coupled to protein by reductive amination using NaBH3CN. [‘H]RAF was coupled first to two relatively long lived plasma proteins, bovine serum albumin and fetuin. The half-lives of these proteins in the rat circulation (&,* = -24 h) was unchanged, suggesting that RAF did not alter the normal mechanisms of protein clearance. When attached to short lived proteins with known sites of catabolism, such as asialofetuin, RNase B, and heatdenatured albumin, neither the tissue nor cellular sites of uptake of the proteins were altered. Thus, 13H]RAFasialofetuin was recovered almost exclusively (\u3e90%) in the liver parenchymal cell fraction, while both [JHJRAF-labeled RNase B and heat-denatured albumin were recovered primarily (\u3e85%) in nonparenchymal cells. In addition, the RAF label was observed to reside stably (tl,2 \u3e 100 h) in the liver following degradation of the carrier protein; in contrast, radioactivity from “‘Ilabeled asialofetuin or RNase B was rapidly (tl,z \u3c 30 min) lost from liver. Radioactivity from 13H]RAF-proteins was recovered in a lysosomally enriched subcellular fraction in liver and consisted of a low molecular weight species (-llOO), containing both glucose and fructose in a ratio similar to that in the original protein. The results of these studies establish that 13H]RAF useful radioactive tracer for detecting the tissue and cellular sites of catabolism of long lived circulating proteins

Role of the Maillard Reaction in Aging of Tissue Proteins: Advanced Glycation End Product-Dependent Increase in Imidazolium Cross-Links in Human Lens Proteins

Dicarbonyl compounds such as glyoxal and methylglyoxal are reactive dicarbonyl intermediates in the nonenzymatic browning and cross-linking of proteins during the Maillard reaction. We describe here the quantification of glyoxal and methylglyoxal-derived imidazolium cross-links in tissue proteins. The imidazolium salt cross-links, glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), were measured by liquid chromatography/mass spectrometry and were present in lens protein at concentrations of 0. 02-0.2 and 0.1-0.8 mmol/mol of lysine, respectively. The lens concentrations of GOLD and MOLD correlated significantly with one another and also increased with lens age. GOLD and MOLD were present at significantly higher concentrations than the fluorescent cross-links pentosidine and dityrosine, identifying them as major Maillard reaction cross-links in lens proteins. Like the N-carboxy-alkyllysines Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine, these cross-links were also detected at lower concentrations in human skin collagen and increased with age in collagen. The presence of GOLD and MOLD in tissue proteins implicates methylglyoxal and glyoxal, either free or protein-bound, as important precursors of protein cross-links formed during Maillard reactions in vivo during aging and in disease