Guanidinylations of albumin decreased binding capacity of hydrophobic metabolites.

AIM: As post-translational modifications of proteins may have an impact on the pathogenesis of diseases such as atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis of these post-translational modifications is established for the clinical diagnostic routine. METHODS: Here, we analysed albumin - the most abundant plasma protein in human - isolated from patients with CKD and healthy controls by chromatographic steps and identified by MALDI mass spectrometry. Post-translational modifications of albumin were identified after digestion by analysing mass signal shifts of albumin peptides using pertinent mass databases. RESULTS: Albumin isolated from plasma of patients with CKD but not from healthy control subjects was specifically post-translationally modified by guanidinylation of lysines at positions 249, 468, 548, 565 and 588. After identification of guanidinylations as post-translational modifications of albumin isolated from patients with CKD, these modifications were quantified by mass spectrometry demonstrating a significant increase in the corresponding mass signal intensities in patients with CKD compared to healthy controls. The relative amount of guanidinylation of lysine at position 468 in patients with CKD was determined as 63 ± 32% (N = 3). Subsequently, we characterized the pathophysiological impact of the post-translational guanidinylation on the binding capacity of albumin for representative hydrophobic metabolic waste products. In vitro guanidinylation of albumin from healthy control subjects caused a decreased binding capacity of albumin in a time-dependent manner. Binding of indoxyl sulphate (protein-bound fraction) decreased from 82 ± 1% of not post-translationally modified albumin to 56 ± 1% after in vitro guanidinylation (P < 0.01), whereas the binding of tryptophan decreased from 20 to 4%. These results are in accordance with the binding of indoxyl sulphate to albumin from healthy control subjects and patients with CKD (88 ± 3 vs. 74 ± 10, P < 0.01). Thus, in vitro post-translational guanidinylation of albumin had a direct effect on the binding capacity of hydrophobic metabolites such as indoxyl sulphate and tryptophan. CONCLUSION: We used a mass spectrometry-based method for the characterization of post-translational modification and demonstrated the pathophysiological impact of a representative post-translational modification of plasma albumin. The data described in this study may help to elucidate the pathophysiological role of protein modifications.

A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus.

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.

Signaling pathways regulating Golgi structure and function.

At the end of the cell division cycle, organelles must be equally distributed to two daughter cells. This includes the many stacks of the Golgi apparatus. Several kinases have been implicated in regulating Golgi disassembly during mitosis, but much about this process remains obscure.