In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Endo-␤-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the ␤1,4 linkage of N,N-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glycoengineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the sitedirected mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K m value of Y217F for an acceptor substrate 4-methylumbelliferyl-␤-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn 175 "knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-␤-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.Endo-␤-N-acetylglucosaminidase (EC 3.2.1.96) (ENGase) 3 catalyzes hydrolysis of the ␤1,4-glycosidic linkage of the N,NЈ-diacetylchitobiose moiety in the core of asparagine-linked glycan of various glycoproteins and glycopeptides. This type of enzyme is widely distributed in animals, plants, fungi, and bacteria. Several bacterial enzymes, such as Endo-H from Streptomyces plicatus (1) and Endo-F 1 from Flavobacterium meningosepticum (2), were cloned and classified into glycoside hydrolase (GH) family 18 in the CAZy data base (available on the World Wide Web), which may share a common evolutional origin with GH18 chitinases. The other ENGases are distinct from the enzymes of the GH18 chitinase family and are classified into the GH family 85. We and others have previously reported that several ENGases of the GH85 family showed significant transglycosylation activity (i.e. the ability to transfer the releasing glycan to an acceptor other than water to form a new glycosidic linkage) (3-6). These ENGases include Endo-M from Mucor hiemalis (3), Endo-A from Arthrobactor protophormiae (4), Endo-CE from Caenorhabditi...

Homogeneous N-glycoproteins carrying defined natural N-glycans are essential for detailed structural and functional studies. The transglycosylation activity of the endo-β-N-acetylglucosaminidases from Arthrobacter protophormiae (Endo-A) and Mucor hiemalis (Endo-M) holds a great potential for glycoprotein synthesis, but the wild type enzymes are not practical for making glycoproteins carrying native N-glycans because of their predominant activity for product hydrolysis. We report in this article the studies on two endoglycosidase-based glycosynthases, EndoM-N175A and EndoA-N171A, and their usefulness for constructing homogeneous N-glycoproteins carrying natural N-glycans. Oligosaccharide oxazoline corresponding to the bi-antennary complex type N-glycan was synthesized and tested with the two glycosynthases. The EndoM-N175A mutant was able to efficiently transfer the complex type glycan oxazoline to a GlcNAc-peptide and GlcNAc-containing ribonuclease to form the corresponding homogeneous glycopeptide/glycoprotein. The EndoA-N171A did not recognize complex type N-glycan oxazoline but could efficient use the high-mannose type glycan oxazoline for transglycosylation. These mutants possess the transglycosylation activity but lack the hydrolytic activity toward the product. Kinetic studies revealed that the dramatically enhanced synthetic efficiency of the EndoA-N171A mutant was due to the significantly reduced hydrolytic activity toward both the Man9GlcNAc oxazoline and the product, as well as its enhanced activity for transglycosylation. Thus, the two mutants described here represent the first endoglycosidase-based glycosynthases enabling a high efficient synthesis of homogeneous natural N-glycoproteins.