Protein O-Linked Mannose ß-1,4-N-Acetylglucosaminyl-transferase 2 (POMGNT2) Is a Gatekeeper Enzyme for Functional Glycosylation of α-Dystroglycan.

Disruption of the O-mannosylation pathway involved in functional glycosylation of α-dystroglycan gives rise to congenital muscular dystrophies. Protein O-linked mannose ß-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2) catalyzes the first step toward the functional matriglycan structure on α-dystroglycan that is responsible for binding extracellular matrix proteins and certain arenaviruses. Alternatively, protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) catalyzes the first step toward other various glycan structures present on α-dystroglycan of unknown function. Here, we demonstrate that POMGNT1 is promiscuous for O-mannosylated peptides, whereas POMGNT2 displays significant primary amino acid selectivity near the site of O-mannosylation. We define a POMGNT2 acceptor motif, conserved among 59 vertebrate species, in α-dystroglycan that when engineered into a POMGNT1-only site is sufficient to convert the O-mannosylated peptide to a substrate for POMGNT2. Additionally, an acceptor glycopeptide is a less efficient substrate for POMGNT2 when two of the conserved amino acids are replaced. These findings begin to define the selectivity of POMGNT2 and suggest that this enzyme functions as a gatekeeper enzyme to prevent the vast majority of O-mannosylated sites on proteins from becoming modified with glycan structures functional for binding laminin globular domain-containing proteins.

Recent advancements in understanding mammalian O-mannosylation.

The post-translational glycosylation of select proteins by O-linked mannose (O-mannose or O-man) is a conserved modification from yeast to humans and has been shown to be necessary for proper development and growth. The most well studied O-mannosylated mammalian protein is α-dystroglycan (α-DG). Hypoglycosylation of α-DG results in varying severities of congenital muscular dystrophies, cancer progression and metastasis, and inhibited entry and infection of certain arenaviruses. Defects in the gene products responsible for post-translational modification of α-DG, primarily glycosyltransferases, are the basis for these diseases. The multitude of clinical phenotypes resulting from defective O-mannosylation highlights the biomedical significance of this unique modification. Elucidation of the various O-mannose biosynthetic pathways is imperative to understanding a broad range of human diseases and for the development of novel therapeutics. In this review, we will focus on recent discoveries delineating the various enzymes, structures and functions associated with O-mannose-initiated glycoproteins. Additionally, we discuss current gaps in our knowledge of mammalian O-mannosylation, discuss the evolution of this pathway, and illustrate the utility and limitations of model systems to study functions of O-mannosylation.

Rapid screening of sugar-nucleotide donor specificities of putative glycosyltransferases.

Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.

Metacognition and Self-Efficacy in Action: How First-Year Students Monitor and Use Self-Coaching to Move Past Metacognitive Discomfort During Problem Solving.

Stronger metacognitive regulation skills and higher self-efficacy are linked to increased academic achievement. Metacognition and self-efficacy have primarily been studied using retrospective methods, but these methods limit access to students' in-the-moment metacognition and self-efficacy. We investigated first-year life science students' metacognition and self-efficacy while they solved challenging problems, and asked: 1) What metacognitive regulation skills are evident when first-year life science students solve problems on their own? and 2) What aspects of learning self-efficacy do first-year life science students reveal when they solve problems on their own? Think-aloud interviews were conducted with 52 first-year life science students across three institutions and analyzed using content analysis. Our results reveal that while first-year life science students plan, monitor, and evaluate when solving challenging problems, they monitor in a myriad of ways. One aspect of self-efficacy, which we call self-coaching, helped students move past the discomfort of monitoring a lack of understanding so they could take action. These verbalizations suggest ways we can encourage students to couple their metacognitive skills and self-efficacy to persist when faced with challenging problems. Based on our findings, we offer recommendations for helping first-year life science students develop and strengthen their metacognition to achieve improved problem-solving performance.

"Oh, that makes sense": Social Metacognition in Small-Group Problem Solving.

Stronger metacognition, or awareness and regulation of thinking, is related to higher academic achievement. Most metacognition research has focused at the level of the individual learner. However, a few studies have shown that students working in small groups can stimulate metacognition in one another, leading to improved learning. Given the increased adoption of interactive group work in life science classrooms, there is a need to study the role of social metacognition, or the awareness and regulation of the thinking of others, in this context. Guided by the frameworks of social metacognition and evidence-based reasoning, we asked: 1) What metacognitive utterances (words, phrases, statements, or questions) do students use during small-group problem solving in an upper-division biology course? 2) Which metacognitive utterances are associated with small groups sharing higher-quality reasoning in an upper-division biology classroom? We used discourse analysis to examine transcripts from two groups of three students during breakout sessions. By coding for metacognition, we identified seven types of metacognitive utterances. By coding for reasoning, we uncovered four categories of metacognitive utterances associated with higher-quality reasoning. We offer suggestions for life science educators interested in promoting social metacognition during small-group problem solving.

Crystal structures of ß-1,4-N-acetylglucosaminyltransferase 2: structural basis for inherited muscular dystrophies.

The canonical O-mannosylation pathway in humans is essential for the functional glycosylation of α-dystroglycan. Disruption of this post-translational modification pathway leads to congenital muscular dystrophies. The first committed step in the construction of a functional matriglycan structure involves the post-translational modification of α-dystroglycan. This is essential for binding extracellular matrix proteins and arenaviruses, and is catalyzed by ß-1,4-N-acetylglucosaminyltransferase 2 (POMGNT2). While another glycosyl transferase, ß-1,4-N-acetylglucosaminyltransferase 1 (POMGNT1), has been shown to be promiscuous in extending O-mannosylated sites, POMGNT2 has been shown to display significant primary amino-acid selectivity near the site of O-mannosylation. Moreover, several single point mutations in POMGNT2 have been identified in patients with assorted dystroglycanopathies such as Walker-Warburg syndrome and limb girdle muscular dystrophy. To gain insight into POMGNT2 function in humans, the enzyme was expressed as a soluble, secreted fusion protein by transient infection of HEK293 suspension cultures. Here, crystal structures of POMGNT2 (amino-acid residues 25-580) with and without UDP bound are reported. Consistent with a novel fold and a unique domain organization, no molecular-replacement model was available and phases were obtained through crystallization of a selenomethionine variant of the enzyme in the same space group. Tetragonal (space group P4212; unit-cell parameters a = b = 129.8, c = 81.6 Å, α = γ = ß = 90°) crystals with UDP bound diffracted to 1.98 Šresolution and contained a single monomer in the asymmetric unit. Orthorhombic (space group P212121; unit-cell parameters a = 142.3, b = 153.9, c = 187.4 Å, α = γ = ß = 90°) crystals were also obtained; they diffracted to 2.57 Šresolution and contained four monomers with differential glycosylation patterns and conformations. These structures provide the first rational basis for an explanation of the loss-of-function mutations and offer significant insights into the mechanics of this important human enzyme.

Advancing the Guidance Debate: Lessons from Educational Psychology and Implications for Biochemistry Learning.

Research in science, technology, engineering, and mathematics education supports a shift from traditional lecturing to evidence-based instruction in college courses, yet it is unknown whether particular evidence-based pedagogies are more effective than others for learning outcomes like problem solving. Research supports three distinct pedagogies: worked examples plus practice, productive failure, and guided inquiry. These approaches vary in the nature and timing of guidance, all while engaging the learner in problem solving. Educational psychologists debate their relative effectiveness, but the approaches have not been directly compared. In this study, we investigated the impact of worked examples plus practice, productive failure, and two forms of guided inquiry (unscaffolded and scaffolded guidance) on student learning of a foundational concept in biochemistry. We compared all four pedagogies for basic knowledge performance and near-transfer problem solving, and productive failure and scaffolded guidance for far-transfer problem solving. We showed that 1) the four pedagogies did not differentially impact basic knowledge performance; 2) worked examples plus practice, productive failure, and scaffolded guidance led to greater near-transfer performance compared with unscaffolded guidance; and 3) productive failure and scaffolded guidance did not differentially impact far-transfer performance. These findings offer insights for researchers and college instructors.

Student difficulties during structure-function problem solving.

Protein structure-function is a key concept in biochemistry. We used the perspective of domain-specific problem-solving to investigate students' solutions to a well-defined protein structure-function problem. We conducted think-aloud interviews with 13 undergraduate students and performed qualitative content analysis to examine the differences in the domain-general and domain-specific knowledge among correct and incorrect solutions. Our work revealed that students used domain-general and domain-specific knowledge in their problem solving. We also identified difficulties for students with the amino acid backbone, amino acid categorization, and causal mechanisms of noncovalent interactions. Using the identified difficulties, we make recommendations for the design of instructional materials targeted to improve protein structure-function problem solving in the biochemistry classroom. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):453-463, 2018.