Review: Modifications of Human Serum Albumin and Their Binding Effect

Abstract

Human serum albumin (HSA) regulates the transport and availability of numerous chemical compounds and molecules in the blood vascular system. While previous HSA research has found that HSA interacts with specific varieties of ligands, new research efforts aim to expand HSA’s ability to interact with more different drugs in order to improve the delivery of various pharmacological drugs. This review will cover fatty acid chain and post-translational modifications of HSA that potentially modulate how HSA interacts with various pharmacological drugs, including glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation.

As one of the most abundant proteins in blood plasma, human serum albumin functions to transport hormones, fatty acids and various compounds through the blood stream. Additionally, human serum albumin also helps maintain osmotic blood pressure.^[1] Human serum albumin has the ability to interact with a variety of ligands, including exogenous pharmacological drugs. Current human serum albumin research aims to enhance albumin’s role from an endogenous ligand transporter to also having a role in facilitating the transport of a variety of exogenous compounds. The ultimately goal is to utilize human serum albumin to improve drug delivery of novel pharmacological approaches to treat various human diseases.

Human serum albumin research has a long history. Its reported that human serum albumin was precipitated from urine as early as 1500 A.D.^[2] Before the 20^th century, non-human serum albumin proteins were already being crystalized.^[3] Clinical use of human serum albumin occurred as early as the 1940’s, when a surgeon by the name of I.S. Ravdin clinically administered purified human serum albumin to seven wounded human patients during the Pearl Harbor attack.^[1] Successfully, all seven patients survived. And then in 1992, Xiao He and Daniel Carter solved the three-dimensional atomic structure of human serum albumin using X-ray crystallography to 2.8 angstroms.^[4]

Human serum albumin is synthesized and secreted from the liver. Human serum albumin is translated from a single gene as preproalbumin.^[5] The protein is imported into endoplasmic reticulum for cleavage of its N-terminal prepropeptide by a serine protease. Afterwards, the protein is transported to the Golgi before eventually being secreted out of the cell. The final molecular weight of human serum albumin is roughly 66,700 daltons.^[6]

Structurally, the human serum albumin protein is mostly composed of α-helices with an overall structure that resembles a heart shape. Human serum albumin has nine double loops spanning three homologous domains.^[7] The domains are named Domain I, II and III. Each domain has two long loops with one shorter loop. The first two loops in each domain are denoted as subdomain A. The remaining loop in each domain forms subdomain B. Thus, human serum albumin has subdomain IA and IB in Domain I, subdomain IIA and IIB in Domain II and subdomain IIIA and IIIB in Domain III. Subdomains with separate helical structures mediate human serum albumin binding with various endogenous and exogenous ligands. However, while the domains have similar structure, each domain has been shown to have different ligand-binding affinities and functions. Two important binding sites on human serum albumin are Sudlow sites I and II. Sudlow site I is located in subdomain IIA and Sudlow site II is located in subdomain IIIA. Sudlow site I has a preferential binding affinity for bulky heterocyclic compounds such as azapropazone, phentylbutazone and warfarin. Sudlow site II seems to preferentially bind to aromatic compounds such as ibuprofen.^[8][9]

Drug interaction with human serum albumin generally enhances the distribution and bioavailability of the drug depending on the specific pharmacokinetic properties of the drug molecules. Additionally, because of its abundance, human serum albumin plays a significant role in the pharmacokinetic behavior of a variety of drugs, including: drug half-life in the bloodstream, regulating drug efficacy, decreasing drug toxicity, and improving drug targeting specificity.^[10]

Human serum albumin-drug interaction can be modified by small molecules such as fatty acid chains. Human serum albumin can directly bind to certain fatty acid chains. Human serum albumin normally interacts with up to two moles of unesterified fatty acids during physiological conditions.^[11] Human serum albumin has seven long-chain fatty acid binding sites throughout its 3 domains. The first five fatty acid binding sites seem to have the amino acid residues to facilitate polar interactions with the charboxylate head of a fatty acid chain. Each site has a different affinity for fatty acids. Fatty acid binding sites #1-5 bind to the carboxylate moiety of fatty acids with electrostatic/polar interactions. Interestingly, increased levels of fatty acid chains such as linoleic acid have been shown to decrease human serum albumin’s binding affinity for sulfonylurea drugs, with gliclazide having the most dissociation from human serum albumin.^[12]

Most drugs that interact with unmodified human serum albumin are anionic, few cationic drugs have been shown to have detectable affinity for human serum albumin. [8] With fatty acid chains, human serum albumin shows increased affinity for cationic drugs. One example of such a fatty acid is myristate, which has been shown to affect human serum albumin-ligand binding.^[13] The laboratory of Hong Liang recently explored the possibility of human serum albumin as a carrier of cationic ligands, using myristate fatty acid chains modifications on human serum albumin at the subdomain IIA to mediate the interaction between cationic ligands and human serum albumin.^[14] Using a combination of fluorescence quenching and X-Ray crystallography, they find that extensive myristoylation of human serum albumin can stabilize the binding of cationic compound amantadine to human serum albumin in subdomain IIA. Further, their data suggests certain anionic drugs can function as potential cationic drug carriers for human serum albumin. While this is still a long way away from reaching clinical settings, this research provides potential method for simultaneous delivery of anionic and cationic drugs using human serum albumin.

Human serum albumin can also undergo post-translational modifications, some of which affect human serum albumin’s ability to interact with drugs. Of all the amino acids, most modifications of human serum albumin seem to be on cysteine 34 in domain I. These modifications include glycation, cysteinylation, S-nitrosylation, S-transnitrosation and S-guanylation (Table 1).

Table 1.

Human Serum Albumin Modifications

Modification	Notable sites	Location
Glycation	Lysine-525 Lysine-199 Lysine-233 Lysine-281 Lysine-438. (Many other glycation sites not listed).
Cysteinylation	Cysteine-34
Nitrosylation	Cysteine-34	(See Above)
Guanylation	Cysteine-34	(See Above)
Dehydralanine	Cysteine-487

Atomic Coordinates were taken from PDB entry 1E78. Illustration of Human Serum Albumin was made with PyMOL.

Human serum albumin can undergo post-translational glycation. Glycation is the binding of a protein with a sugar molecule without the assistance of an enzyme.^[15] On human serum albumin, these sites include Arginine 114, 218 and 428 as well as Lysine 186.^[16] Glycation has been reported to alter human serum albumin structure.^[17] Glycation of human serum albumin seems to affect the binding of some, but not all drugs. While initial reports suggest that glycation may affect human serum albumin’s binding ability to drugs such as warfarin, later publications did not find this significant change.^{[18,19,20,21]} Other compounds such as tolazamide, acetohexamide, glibenclamide, and tolbutamide have at least a 40% decreased binding affinity to glycated human serum albumin compared to non-glycated human serum albumin.^[22,23] Glycated human serum albumin has been shown to have five-fold increase in binding activity to L-tryptophan compared to non-glycated human serum albumin.^[24] While increased glycation of human serum albumin is usually correlated with increased blood glucose levels, as seen in diabetic patients, a recent in vitro study suggests that Zinc concentrations may also regulate human serum albumin glycation.^[25] Additionally, glycated human serum albumin has drawn recent interest as a supplementary monitor for monitoring glycemic levels in diabetic patients. While blood glucose provides an immediate and transient measurement of blood glucose at the time of measurement, glycated human serum albumin stays present in the bloodstream for 2-3 weeks.^[26] Therefore, tools measuring glycated human serum albumin may function as better monitors for glycemic levels over longer periods of time.

Human serum albumin can also undergo cysteinylation, or the addition of another cysteine to Cysteine-34 on human serum albumin via a disulfide bond.^[27] This modification has been suggested to be facilitated by cystathionine β-synthase (CBS), as CBS deficient mice lack cysteinylated human serum albumin.^[28] This modification was found in patients with liver and kidney diseases, as well as patients with diabetes. Cysteinylated human serum albumin levels correlate with high-risk pregnancies and uteroplacental insufficiency (UPI), suggesting that measuring cysteinlyated human serum albumin levels may be advantageous to monitor pregnancies affected by UPI.^[29] Increased human serum albumin cysteinylation has also been observed at the end stage of renal disease patients.^[30] While several studies have elucidated the potential role of cysteinylated human serum albumin in patients with diseases, little is known about how cysteinylation may affect human serum albumin’s ability to interact with ligands. One study from the researchers of the Maruyama lab find human serum albumin’s binding affinity to bilirubin and tryptophan, as well as drugs such as warfarin and diazepam, was significantly decreased.^[31]

Cysteine residues on human serum albumin can also undergo S-Nitrosylation or incorporation of nitric oxide.^[32] While nitrosating agents such sodium nitroprusside have low reactivity to the sulfhydryl groups in human serum albumin, S-nitrosoglutathione intermediates seem to interact with the sulfhydryl groups of albumin and facilitate S-nitrosylation.^[33] Currently, it is unclear how S-nitrosylation affects the binding of human serum albumin to its ligands. Nonetheless, a few papers offer some clues. S-nitrosylation of the Cys-34 on human serum albumin increases its affinity for Copper (II) and penolsulfophthalein (or PSP), both of which are normally circulated in the blood. This suggests S-nitrosylation may regulate the transport of organic anionic compounds and heavy metals through the circulatory system.^[34] A more recent paper published in 2008 by the Otagiri lab shows that S-nitrosylation of human serum albumin affects its ability to interact with fatty acids, specifically oleic acid.^[35] Mechanistically, they show when oleic acid binds to human serum albumin, it leads to greater accessibility of a single thiol group on albumin. They also conclude oleic acid fatty acid binding increases S-denitrosation and S-transnitrosation of S-nitrosylated human serum albumin. In addition to oleic acid, they find S-nitrosylated human serum albumin also strongly interacts with Bilirubin and weakly interacts with L-tryptophan, progesterone, ascorbate, Zinc, and iron.^[36] Besides Cys-34, Cys-410 has also been identified to undergo S-nitrosylation.^[35] Lastly, nitric oxide itself is a chemical messenger which is regulated by human serum albumin. The Nudler research laboratory shows that human serum albumin interacts with S-nitrosothiols, functioning as a nitric oxide sink to ultimately increase the formation of low-molecular weight S-nitrosothiols in order to regulate blood pressure.^[37]

Some new post-translational modifications of human serum albumin include S-guanylation and dehydroalanine conversion. S-guanylation of cysteine 34 is a recently reported modification of human serum albumin discovered when comparing blood samples between healthy patients and hemodialysis patients.^[38] S-guanylation modification occurs when an 8-nitroguanosine 3′,5′-cyclic monophosphate group reacts with sulfhydryl groups of human serum albumin. While it is unclear how this modification may affect drug binding, research suggests that this protein may function as a endogenous antibacterial agent. Only minor structural conformational changes were observed with this modification. Dehydroalanine conversion is another recent post translational modification found on human serum albumin. Previously found in in vitro environments, cysteine residue conversion to dehydroalanine was found present in human patient blood plasma when healthy blood samples were compared to critically ill patient blood samples. However, it remains to be seen whether this modification significantly affects human serum albumin’s biological function and its ability to interact with exogenous drugs.^[27]

In conclusion, while human serum albumin is a single non-glycosylated protein, accumulating research shows that more residues on human serum albumin can undergo certain post-translational modification in specific environments. Human serum albumin can undergo a variety of modifications including: glycation, S-nitrosylation, S-guanylation, and dehydroalanine conversion. Current research shows that some of these modifications affect human serum albumin’s ability to interact with exogenous drugs. Additionally, several reports show that fatty acids can impact how these post-translational modifications can regulate human serum albumin binding ability. Human serum albumin has an established role as a blood stream carrier, it will be interesting and exciting to test how these modifications to human serum albumin can affect and potentially help the drug delivery of various pharmacological treatments for different diseases.

Abbreviations

HSA

Human serum albumin

MYR

Myristate

FA

Fatty acids