It is well known that dogs suffer from at least two unusual traits related to Cu metabolism, which are (a) an inability of their serum albumin to bind Cu tightly, due to no histidine in the N terminal binding site; and (b) 10–20‐times higher liver Cu concentrations accompanied by a tendency to develop Cu overload.[1,2] We have identified and investigated two additional peculiarities in dogs related to components in their blood plasma. One has to do with ceruloplasmin (Cp), a 132 kDa glycoprotein, which has functions ranging from delivery of Cu to cells and scavenging reactive oxygen species, to oxidation of Fe(II) and various amines. It is also the main Cu binding component of the blood plasma. Although liver Cu concentrations are extremely high compared to other species, total Cu levels in plasma are normally much lower than those in human and rats (200–400 ng/ml versus about 1200). We found that when the plasma of Labrador retrievers was fractionated in large pore size exclusion chromatography (SEC), the main Cu peak eluted much earlier than is the case with plasma from other mammals. In Superdex 200 FPLC equilibrated with 20 mM phosphate, pH 7, the main Cu peak eluted near the void volume (Mr ~ 700 kDa) as compared to that of humans mice, rats and pigs (Mr~150 kDa). The same was the case for plasma from other dog breeds. Western blotting determined that Cp protein eluted in parallel with the main Cu peak and was of normal size in SDS‐PAGE. Enzyme activities associated with Cp (ferroxidase and p‐phenylenediamine oxidase) also eluted in parallel. This indicated that canine Cp was either bound to some other protein(s) or that it was aggregating. Lactoferrin, which has been reported to bind to Cp in milk, was not detected. Canine plasma proteins were further fractionated on Sephacryl S300, which gives a better separation of large components. Stained SDS‐PAGE gels of fractions from such columns did not show any bands that eluted in parallel with those of Cp. This is consistent with the concept that Cp was not binding another protein but aggregating. To further examine that possibility, fractionation was carried out in buffers of higher ionic strength. Isotonic phosphate‐buffered saline did not alter the elution of canine Cp in Superdex 200. However with 300 mM phosphate (pH 6.8), canine Cp eluted in the same way as the Cp of other mammals, indicating it had dissociated. The other canine peculiarity investigated relates to the tendency towards Cu toxicosis. Plasma from Labrador retrievers with mutations in the Cu transporter ATP7B and high liver copper concentrations had markedly increased levels of a small Cu carrier in their plasma and urine compared with the wild type, as is the case with Wilson disease model mice.[3] Our results indicate that increased levels of a small Cu carrier may be helpful in diagnosis of canine Cu overload, and suggest that canine Cp is circulating as a multimeric protein.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The main route for copper excretion in mammals is via the bile, little exiting through urine. In Wilson disease, biliary excretion is prevented by lack of an active form of ATP7B, which permits copper entry into bile. This leads to copper accumulation and severe liver toxicosis. Dogs tend to have the same problem, with much higher liver copper levels than other mammals. Many die of liver copper toxicosis. As in Wilson disease, Atp7b‐/‐ mice excrete high levels of copper in urine. Excretion is in the form of a 1.5 kDa SCC. We now have evidence that the same SCC exists in the blood plasma of humans, mice and dogs. Normally, it is bound to larger proteins, but large amounts of free SCC occur in the plasma of Atp7b‐/‐ mice, and in some dogs. SCC can be released from larger proteins by EDTA and accounts for ~10% of the total copper in human plasma. SCC is also released by EDTA in plasma from dogs and mice, including those with “free” SCC. FPLC‐SEC preliminary data suggest SCC is riding on proteins >200kDa. Dog ceruloplasmin is also unusual, eluting as a much larger protein, although of normal size in SDS‐PAGE. We conclude that SCC may offer an alternative route for copper excretion that can be mobilized in some mammals in response to liver copper accumulation, and that the study of dog plasma may reveal additional information about copper metabolism not detected as easily in other species.
We recently discovered a small copper carrier (SCC), which in most mammals is mainly bound to larger proteins and can be released by EDTA. It normally accounts for at least 10% of total plasma copper, and more than half the total in Atp7b−/− mice and some Labrador retrievers with copper overload, where it circulates in the “free” form and appears in the urine. This suggests SCC is an alternative means of excreting copper when biliary excretion is inhibited. To begin to isolate and characterize SCC, blood plasma of human volunteers (permitted by the university IRB), pigs, cows, sheep and Labrador retriever dogs were analyzed for their quantities or free and protein‐bound SCC, using 10 and 3kDa ultrafiltration and size exclusion chromatography (SEC) with Superdex 200 and peptide FPLC. By MALDI‐TOF, the main component identified as potentially SCC consistently had a mass of 1329.5 Da (not including Cu), identical to that reported for urinary SCC from Atp7b−/− mice. It eluted between vitamin B12 and NADH in small pore SEC and not with Cu‐EDTA, which aggregated. Two amino acids were identified; a third was blocked by sulfate or phosphate. However, solution NMR indicated the samples were not pure. Preliminary data suggested phenyl HIC and anion exchange might be useful purification steps. Plasma from the pig had the highest levels of total SCC and more than 10% in the “free form”. So it was chosen to identify the best steps for large scale SCC purification.
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