O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

Inappropriate accumulation of the misfolded Z variant of alpha1-antitrypsin in the hepatocyte endoplasmic reticulum (ER) is a risk factor for the development of end-stage liver disease. However, the genetic and environmental factors that contribute to its etiology are poorly understood. ER mannosidase I (ERManI) is a quality control factor that plays a critical role in the sorting and targeting of misfolded glycoproteins for proteasome-mediated degradation. In this study, we tested whether genetic variations in the human ERManI gene influence the age at onset of end-stage liver disease in patients homozygous for the Z allele (ZZ). We sequenced all 13 exons in a group of unrelated Caucasian ZZ transplant recipients with different age at onset of the end-stage liver disease. Homozygosity for the minor A allele at 2484G/A (refSNP ID number rs4567) in the 3′-untranslated region was prevalent in the infant ZZ patients. Functional studies indicated that rs4567(A), but not rs4567(G), suppresses ERManI translation under ER stress conditions. Conclusion: These findings suggest that the identified single-nucleotide polymorphism can accelerate the onset of the end-stage liver disease associated with alpha1-antitrypsin deficiency and underscore the contribution of biosynthetic quality control as a modifier of genetic disease. (Hepatology 2009.)

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation. To distinguish the intracellular site where the glycoprotein ERAD signal is generated, a COPI-binding motif was appended to the N terminus of the recombinant protein to facilitate its retrograde translocation back to the ER. Partial redistribution of the modified ERManI was observed along with an accelerated rate at which N-linked glycans of misfolded α1-antitrypsin variant NHK were trimmed. Despite these observations, the rate of NHK degradation was not accelerated, implicating the Golgi complex as the site for glycoprotein ERAD substrate tagging. Taken together, these data provide a potential mechanistic explanation for the spatial separation by which glycoprotein quality control components operate in mammalian cells.

Endoplasmic reticulum (ER) α-1, 2-mannosidase (ERManI) contributes to ER-associated protein degradation (ERAD) by initiating the formation of degradation signals on misfolded N-linked glycoproteins. Despite its inferred intracellular location, we recently discovered that the mammalian homologue is actually localized to the Golgi complex. In the present study, the functional role of Golgi-situated ERManI was investigated. Mass spectrometry analysis and coimmunoprecipitation (co-IP) identified a direct interaction between ERManI and γ-COP, the gamma subunit of coat protein complex I (COPI) that is responsible for Golgi-to-ER retrograde cargo transport. The functional relationship was validated by the requirement of both ERManI and γ-COP to support efficient intracellular clearance of the classical ERAD substrate, null Hong Kong (NHK). In addition, site-directed mutagenesis of suspected γ-COP–binding motifs in the cytoplasmic tail of ERManI was sufficient to disrupt the physical interaction and ablate NHK degradation. Moreover, a physical interaction between NHK, ERManI, and γ-COP was identified by co-IP and Western blotting. RNA interference–mediated knockdown of γ-COP enhanced the association between ERManI and NHK, while diminishing the efficiency of ERAD. Based on these findings, a model is proposed in which ERManI and γ-COP contribute to a Golgi-based quality control module that facilitates the retrieval of captured ERAD substrates back to the ER.

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive. Herein, the results of transient expression, RNAi-mediated knockdown and functional studies demonstrate that the transcriptional elevation of EDEM1 boosts the efficiency of glycoprotein ERAD through the formation of a complex that suppresses the proteolytic downregulation of ER mannosidase I (ERManI). The results of site-directed mutagenesis indicate that this capacity does not require that EDEM1 possess inherent mannosidase activity. A model is proposed in which ERManI, by functioning as a downstream effector target of EDEM1, represents a checkpoint activation paradigm by which the mammalian UPR coordinates the boosting of ERAD.

Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes.