N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 78

↔ N -Hcy-SH-transferrin + albumin-Cys 34 -S-S-Cys

↔ N -(Hcy-S-S-Cys)-transferrin + albumin-Cys 34 -SH

Reaction 5.5 Thiol-disulfide exchange between N-Hcy-transferrin and albumin-Cys 34 -S-S-Cys.
Similar reactions occur with other N-Hcy-proteins, such as N-Hcy-fibrinogen, N-Hcy-antitrypsin,
N-Hcy-hemoglobin, N-Hcy-myoglobin, and N-Hcy-cytochrome c

Hcy-SH

S-S-Cys S-S-Cys

H 2 N HN

Lys 525 Cys 34

+ Hc y-thiolac tone

Cys 34

Lys 525

Hcy-S-S-Cys

HN SH

Lys 525

Cys 34

Hcy-SH

H 2 N

SH HN SH

+ H cy-thiolac tone

Lys 525 Cys 34 Lys 525

Cys 34

Fig. 5.9 N-Homocysteinylation of Lys 525 prevents the structural transition in albumin dependent
on the status of the conserved Cys 34 residue (Reproduced from [69])

Of the seven lysine residues of human albumin (Lys 4 ,Lys 12 ,Lys 137 ,Lys 159 ,Lys 205 ,
Lys 212 ,andLys 525 ) identified as targets for N-homocysteinylation by Hcy-thiolactone
in vitro, Lys 525 is a predominant site of N-homocysteinylation, while Lys 137 and Lys 212
are minor sites, both in vitro and in vivo (Fig. 5.2 ) [212, 213]. Taken together, these
results provide evidence for a novel form of albumin, N-(Hcy-S-S-Cys)-albumin-
Cys 34 -SH (Figs. 5.7 and 5.9 ), and suggest that a disulfide at Cys 34 , a conserved residue
in albumins from various organisms, promotes the conversion of N-(Hcy-SH)-albumin-
Cys 34 -SH to a more proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys 34 -
SH, which would facilitate clearance of the N-homocysteinylated form of
mercaptoalbumin. These data also suggest that, by rendering Cys 34 reduced, N-
homocysteinylation prevents the structural transition in albumin dependent on the
status of the conserved Cys 34 residue (Fig. 5.9 ).
N-Hcy-albumin becomes more susceptible than native albumin to aggregation
[78] and oxidative damage by hydrogen peroxide [96]. Exposure to N-Hcy-albumin
increases monocyte adhesion to the endothelial monolayers in a co-culture model
[169]. Unmodified albumin does not have any effect on monocyte adhesion. The
increased adhesion is observed only when N-Hcy-albumin is used at concentration
observed in hyperhomocysteinemic subjects, but not when the concentration
corresponds to that observed in normal individuals. The increased cell adhesion is
accompanied by upregulation of genes involved in the inflammatory response
(ICAM-1, VCAM-1) and vascular remodeling (ADAM17, MCP1, Hsp60) in both
endothelial cells and monocytes. N-Hcy-albumin also induces release of Tnf- from

endothelial cells to the medium. As these responses are observed at relatively low
concentration of N-Hcy-albumin, these findings suggest that N-Hcy-protein rather

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