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>> good afternoon. for those, some of you who have not been here before, that's the brooklyn bridge which is the logo of this activity. it's intended to mean that we're connecting advances in basic biological and engineering sciences with human health and
medicine. and we the people on the cat walk halfway between brooklyn and manhatten. and the important thing is that once you build something like this, life is never the same on either side. wonderful things happen.
and the same thing happens when people of different disciplines sit down, have coffee, talk, and learn the other person's language. that's the whole intent of trying to demystify, maybe we should call the course linguistics 1a or something like
that. so in an important announcement here. i just found out today, and this is more for the people who listening and watching, usually 2-400 a week just on the nih campus. previously, these sessions were
live only within the nih environment, your computer. but it tissue turns out all these sessions can be viewed anywhere on earth or space by just going to video cast nih.gov . that's live. and then one or two weeks later,
all the sessions are put up on y outube and the nih video ar chive. for the whole 12 years that we have been doing this. so you can access it. most of our sessions are actual ly accessed, the numbers get into the several thousands.
so we must be doing something right. in this, the program for this year, there is sort of a background to -- about five of the sessions, and the background is inflammation. to set on fire. and way back in the first
century, infectious inflammation , abscesses, boils, infected wounds led to the classic description that it's characterized by readiness, swelling, pain and loss of function. in the 21st century, inflammation has now broadened
its scope. in fact, it's hard to find any entity from a clinical perspective in which inflammation is not involved. so several of the sessions that we will continue to have during the year concern the role of inflammation and other disease
processes which have not conventionally been identified as associated withnationinous as a major determinant of the phenotype. so last week, we heard about cardiovascular disease. from dr. gallin. this week allergies are one of
the main topics that will be brought up. and you'll see, in february, immunosuppressive diseases, hiv, cancer, fibrosis, autoimmune, neuro dejenningsive, obs, it's almost as if every area of clinical medicine has an important component related to
inflammation and to the complex inflammatory cycles that take place, as well as the host responses in terms of the com plexities of the immune system. so today we're going to consider something that -- for an area which four many years can be
said to have had somewhat of a prolonged infancy, i think. but has now progressed well into and beyond adolescents, truly an explosive area of biomedical research and important clinical findings. into the big umbrella of cli combics. -- gly combics.
it's obvious that the genome, the sequence, has an answer to everything and it raises many more questions. epigenetics is certainly one of the major aspects of complications. and today's post translational modifications of glycoproteins
and glycolipid turn out to be critical in many, many functions , in regulation and disease. there is a link to inflammation which we will hear about, squeezes are both inheritbling and acquired. so we have 3 speakers today.
i'm going to briefly introduce them all to you. the best tennis player of the lot is john hanover, who is chief of the laboratory of cell and molecular biology around nid dk, received his ph.d. from hopkins and has been here since 1981 when esa post cookeral
fellow. he is truly one of the major movers in enhancing the recognition of glycoprotein biology and pathobiology. and our second speaker is michelle bond, who received her ph.d. in organic chemistry from stanford, a staff scientist
in the niddk in dr. hanover's area. the third is jonathan lyons. jonathan? where did you go to medical school? university of southern california, i'm sorry, i didn't see it in the thing here.
okay. john is a clinical investigator in the laboratory section of the allergic diseases in niaid. he's been interested in studying the consequences of altered glycosylation in allergy and agent tropy. we're very grateful to the three
of you for being willing to spend a couple of hours and ex citing us about important area where you played such a critical part. >> hello everyone. good afternoon. i'm john hanover, a glyco biologist.
no, it's not a 12 step program. you don't have to say hi john. i'm how many ted talk fans do we have here? so for those not familiar with the genre, this someone, an engaging speaker, speaks in a contrarianian voice about something you think you know
without but you don't. i'm going to do just the opposite of a ted talk. i'm going to talk about something that i think no really cares to know about when they're training, but then later on in their training they realize, i should have been paying
attention when they talked about carbohydrate metabolism. i hope we can convince you of that kay. in my view, glycans are really -- the origin of life are really glycans that have develop ed the ability to self- rope pliicate.
and then propagate to the most a bundant bio molecules on earth. it's a lot to benisted in. with regard to human disease, i want to start with this slide which gives, i think, a really tribute to the predecessors, my predecessors and others in in the intramural program.
this is one of the hubs, particularly in glycoscience for the past 50 years. the work of carbohydrate chemist hudson and fletcher and the work of the esteemed glycobiologists shown before. i had the good fortunate of working in and around of all
these folks. i want to also mention the glyco science interest group. if we capture your attention today on may 5, on this campus, there will be what is known as glyco-day. a wonderful meeting with glyco biologists if.
it's a wonderful way to spend a day, may 5 this year. i want to give a nod to the un diagnosed disease program, they are an important part of john works closely with them. he'll have more to say about that at the end. this so have is quote from my
creeing, bill. i like to say the rare diseases provide rare insights. and the way bill expresses this, here, he said people with rare diseases give humanity so much scientifically and spiritually that we owe them a huge debt of gratitude.
they make us more human. i'm really pleased that this community as embraced the rare disease program and, as you know , there is a rare disease program day every year on the nih campus. so my intersection with what we're going to talk about really
started a few years ago. and this individual and his wife were trying to identify the disease under lying their daughter grace's affliction. she went to -- he went to a number of clinics with very little -- very little insight until he approached a genomicist
, willing to perform exon sequencing for grace. the long story short, grace was actually born with a deficiency in in an enzyme that i had blundered upon. i use that word directly, blundered upon, as a graduate student in the 1970s.
that enzyme cleaves the glycosylation chain in the cytoplasm off proteins that are retro translocated from the er. at about the same time, matthew might shown here meeting with our former president, president obama, on -- this was the press conference regarding precision
medicine, had a daughter also effected. in fact, sorry, his son was and his son is shown here. and those two drivers have been advocates of patient -- these really is patient advocates for rare diseases. and these 2 gentlemen, the 22
matthews, have -- two matthews, have pushed awareness of rare diseases. john made this deficiency, a very rare disorder and considered the first of the dis ordered of gly solelation associated with carbohydrate re moval.
we are actively working on the problem. today, though, we want to focus on the role of glycans in physiology, particularly related to the immune system. after a brief overview that i'll give, i'd like to previously touch on glycoprotein bio
genesis as a field. and then michelle will give a little bit more detail on the nucleotide surings, why we think they're under studied and important area of research. then john will talk more clinically about pgm3 deficiency , tied to nucleotide
metabolism and carbohydrate met met. so let me start by telling you that carbohydrates and their recognition are important for all aspects of cell physiology. no more so -- this is no more apparent than in the immune system where glycos are involved
in so many aspects it's hard to innumerate them. in inmate immunity through recognition of carbohydrates on pathogen cell surface. activation of immune cell. trafficking. across the called -- the neutrophil migration.
the immune cells and the pro duction of antibodies and antibody structure. so it's -- no aspect of immune roll that isn't touched by glycan. when we talk about glycans ironically, we're talking about a very broad class of bio
molecular. some shown here. i'm going to be focusing most of the discussion today on the n glycans shown here, i in part because they represent the most abundant disorders but you can see here that the o glycans, other o glycans such as though
modifying notch, gpi, which are quite abundant, and various form s of glycans, very, very long, non template driven chains that are acidic that sequestration nearly every growth factor important for cell activation. that is a background.
let me tell you why glyco biologists have been recruited into many areas of modern pathophysiology. the rarity and severity of genetic diseases highlight the importance of this. these congenital disorders are typically less than 1 in 20,000
in the u.s. population, yet, they reveal principles that are applicable to other chronic human diseases. some examples of these diseases are shown here. this is by no means exhaustive, the defective o glycollation seen in maryland -- md.
you'll hear more about this lat er. notch signaling, mediated by glycans. the selectens, one of the key players in the inflammation response. galectins, play a key role in recognition and immunity.
the proteoglycans themselves, se questering growth factors being involved in morpho genesis are major targets. we'll talk about those in a moment. most glycans -- glycans play a key role in the act of bactera to hone to the surface on the
bacterial surfaces in our gut, so-called microbiome is dictated by glycans. heparin, this drug is actually a gag. people don't think of it that way. it's glucose mean glycan. people continue reelingize it's
a gag. there are also many monoclonal therapeutics and glycoforms of these are quite important. cancer biomarkers is an extreme ly important growth area. and finally, the vaccines to infectious organisms, most are actually targeted to glycan.
so woe can't ignore this area bio medically. so i want to kind of extend what-- talking about the glyco genome. even though it's under-studied it represents about 5% of the human genome. 5% of the human genome encodes enzymes that act on carbohydrate
s. 2% encodes glycocell transferase that's an enormous expenditure of energy to make this post transsational modification. the. >> anchor: genome, being -- glycogenome being different from what we think of normally is
actually mediated by mod ifications of the encoded proteome. so the transcript ohm encloudy skies the proteome, acted by transferase, to create this vast assembly of complex molecules that represents the so-called this is true in the lipids.
these two mols represent non template driven diversity and inflammation. an example of how this works is shown here. a nuclei nucleotide donor to a protein receptor. these are template driven in the sense that one precursor has to
be made before it can be ex tended. by separating the enzymes of -- in different subject set of compartments you can organize the assembly of the pathway. we'll talk about how that happen s, how it's important and how it can be deregulated.
so i may be the only glyco biologist in the country who has is head of a genomics core facility. and don't ask me how that happen ed. but one of the remarkable things that's happened inscience in the last few year years is the
tremendous explosion through sequencing of exome sequences. so exome and genome sequencing has greatly accelerated, not just the discovery of the congenital disordered but other fairly rare disorders. the genome project fueled the growth of the carbohydrate
cannot aing enzymes that we know about structurally, came from the genome project, particularly , the microbiome project. exome sequencing -- i like this graph. does every one know what more's law is?
it came about as the result of the transistor industry. it was noted the number of circuits you could print on transistors followed this kind of law, that is the cost dropped precipitously with time. up until this date, 2008, sequencing follows more's law.
at that point, new jen sequence ing caused a dramatic drop in the cost per genome. it's not exactly free but the human john costs have dropped. so estimates suggest that with current -- the 10,000 genomes we have available to examine, the 20%, 20% of the population,
african, and european american, after congenital disorder. >> i coslation wheel. if we look at the rates of detection, the ability to see both carrier mutations and the ability to diagnose diseases that appear in young children, particularly to a paediatric
screening, is dramatically increased. now, john is going to talk about this in much greater detail. i wanted to give a historical reminiscence about this. the way this field has progress ed, the young folks who present with the congenital dis
orders have a few common features, hypotonia, being perhaps the most obvious. when people bring these young kids to the pediatrician, it is not always the pediatrician who diagnoses these children. and so i want to show how these are typically diagnosed.
for many years, bio chemical measures were used to screen these kids. and in a recent review able these were listed with appropriate references. i can give you the references if you want to follow up on that. in general, people have followed
the disorder by followng serum proteins like tran feren, and the electric fer ret mobility of those. for gpi there is a series of markers, there are some enzyme markers, some better than others , that allow partial diagnosis of gpi anchors, john
will elaborate on this later. for the alpha glycans, there is a good antibody that allows the diagnosis of this. this has been used clinically now for at least 15 year years to my knowledge. so what are we talking about? with these diseases, the most aa
bundant, by far, are those in glycans, in the gly solelation. about 40 gene products, 40, are involved in the assembly of this structure. so when you about that that's a tremendous amount of energy to put in a structure so highly cnserved.
i'll show you in a moment, it's absolutely essential for cell ular proteostasis. for the folding reaction and the proper quality control in the er second most abundant -- glycans, we discussed these. made of acidic residues typically hedonic acid.
the gpi anchored proteins. then a series of svgs associated with syntheses, in particular, those associated with the musn't type glycans and others on molecules like notch. so i'm going to focus on glycosylation. what do we know?
we know that throughout metazooa this shown here is a symbol of a step-wise pattern on the lipid anchor. the lipid anchor is a -- this is built up with 3 glucose residues , then trimmed down on on on gokey. how cue we know this?
we learn it from many years of bio chemistry, inhibitors of the type i'm showing here. some of these have been used clinically to inhibit these pathways. we've learned a lot from yeast and somatic cell genetics, and perhaps most importantly for
today's discussion, from human congenital disorders of many pathways were first described as congenital consist orders, very interesting. so this is one field where human diseases are really instructive of the biochemistry and genetics the most common form of cvg
effects this n link glycosylation pathway. there is about 40 gene products. this precursor assembled largely within the lumen of the endo plasm -- although the initial steps are built up on the cytoplastic surface. so while we tend to think of
glycosylation of being restrict ed to the endomembrane system, the initial steps of this pathway are cytoplasic events. that was what i did my thesis on many years ago when it matters. so why do we go through this elaborate multi step process?
it turns out glycosylation represents one of the key quality control mechanisms. two diseases i would like to highlight. the unblocked transfer of the lipid, poly peptide chain, occur s in the er, cotranslation ally or immediately
after translation on the ribo some. the reason that's important is because you need to add a big bulky sugar in the kinetically limited time, in a kinetic -- physically limited space. you're adding as many as 14 glucose and mannose read dues
all in about -- it's been calculated, 30-40 milliseconds. that can only be done by an activated nucleotide sugar. couldn't be done step-wise. upon addition that protein is, then, subject to retention by a series of lactosens, hold it in the er until folding occurs.
when proper folding occurs, it's released and allowed to be moved through the secretary pathway. however, there is something called a manos timer which allow s this man notices timer, monitors the quality of the protein, the text regions of mis folded disorder.
it binds to the lectin, can't leave the er. i don't know if you remember watching lucy, the lucy show where lucy andethal are in the assembly line with the chocolate and they're sitting there, as the chocolates go by, originally they can keep up.
all of a sudden, my god, they're throwing chocolates in their mouth. it's horrible. you have to see it if you hadn't this is a lot like that. if you screw up this basic homeo static mechanism, folding goes awry.
we know that because in deficiency is a disease of protein folding, that is, ngly1, removing this glycan after it's translocated out of the ere really screws up degradation of protein. so it depends on removing this chain resulting from misfolding.
another disease for those who heard [indiscernible] talk last year, is the deficiency involved in early stages of inglycan synthesis. if you don't -- if you don't carry out these initial steps in the glucoselation reaction, you have a protein folding.
just to complete this story, there are a lot of congenital disorders of glycosylation at a occur in the er. the gogi is an elaborate processing of this structure. if you go to a meeting there is still a lot of interest in how all of these enzymes are
regulated. it's a little bit like a chorus, like the mormon tabernacle we're choir. you have the sopranos, altos. every one is singing but they need to harman harmonize. -- they need to harnenize. if we look by compartment, you
can see that in the cytosol, we're going to be talking -- michelle will tell us more about this, the pathway, the er representative of these, there is one -- i think there is more than one now known. and finally, there are some 20 enzymes that are deregulated in
the gogi. with that, i'm going to turn the podium to my younger more cap able colleagues. enough of my ranting. we'll talk about some work we're doing jointly to understand one of these diseases and how we approach it compartmentally.
so i'll invite me colleague, michelle bond, to continue the discussion. >> thank you. and i'm going to spend the next few minutes talking more about the molecular details about what john was talking about in his slide deck.
and what i'm going to argue is that being able to understand exactly what's happening on both a biological and chemical level for all the pathways required for glycan synthesis will be incredibly important for understanding the molecular de tails of the diseases that can-- general disorders of gly
coslation. some of that lies in the fact that these glycans are incredibly complex, so they can be very large and they're dynamically altered. so john talked about n glycan. i'm going to talk about one particular sugar modification
toward the end of my talk. but what do we start with? we have a prepare represented by the circle. then we have a seggar appended to it. that sugar could be something as complex as this n link glycan here which is attached to an a
passengering rescue, or simple. so one of the things i wanted to point out that's in common for both of these glycans is the fact that glue seem mean, the blue sugar, is common for both. interestingly, incredibly importantly, udb, sugar in you cleo tied and glucose mean are
common for the past majority of glycans that the cell sin that sizes. how do we get to either of these ultimate -- to these sugars being placed on glycans? we have to have a conoutrage the receptor. and we can't have a sugar that
not -- not high energy molecule due to the fact that the sugar wouldn't have any sort of leav ing group for the transferase to be able to utilize. so we have a glycoconjugate we have a sugar donor which i have written as ndp.
and the -- we have to make sure they're at the right place in the right time at the right concentrations for all these reactions to take place effect ively. and some of those are also nucleotide sugar transporters. and glycan remodeling instills
which i don't think we'll spend time talking about today. so i wanted to talk about what exactly is a nucleotide sugar. what i have here is the structure of [indiscernible]. i've elected to use udp as -- this is an important theme throughout, what i'm talking
about and what john is going to talk about in his slides as well so we have 2 important components of the nucleotide sugar. we have phosphate, and we have the sugar portion on the left in blue which is glucos mean. sugar is analba linkage to these
groups and this is activated molecule. and what do i mean? well, you're probably much more familiar with atp, and this is also a very active molecular. so all of the negative charges that you see both the udp, and the atp, are mutually repulsive.
so the non reducing terminus of one of these large ligands that john was talking about can at tack the position of -- this sugar right here. meaning that the udp will be a good group. this is something that couldn't happen about this molecule being
in tact. so how do we get to the synthesis of this ndp sugar? we have a sugar that is either scavengeed from degradation products within the cell or nutrients from the extra cellular milieu. and it's phosphorylated at the 6
position. this is incredibly step because this strap traps the sugar within the cell. the suggest irrelevant -- the sugar can no longer diffuse. finally, the sugar one phosphate is converted to the ndp sugar. this is fairly uif i cantis for the production of all these
nucleotides. and that's a fairly simplistic view. what i'd like to talk about is the integreated process required for this synthesis of multiple sugar nucleotide sugars. you'll notice that on the slide, we have 8 of the 9 sugar
building blocks. so galactose does, glucose, fructose, if you does, manos, and acid. and in most cases, we can go from an individual sugar mono saccharide and several steps to the production of that nucleotide sugar.
what's really interesting, and the reason that udp in the middle, is not by accident. it's importantly integrated in the synthesis of multi sugars. so you can see that glucose can be converted to 6 phosphate, which in several steps can be converted to glcnac, being a
component of udp, so this is termed the bio synthetic pathway there are other inputs for this, starting with glcnac itself. one other thing that is key to note, all of these pathways have rate limiting enzymes which are in the red boxes. and those enzymes in some cases
can also be feedback inhibited by the ultimate product of their pathway. so for example, g pat here can be feedback inhibited by udfglcn ik. if there is an overabundance, the bio synthetic pathway is going to stop producing this
product. the other important thing to note is that here i've starred the different enzyme that are -- that are actually disease associated. and i think this is critical to crimea given the concentration of these components is
incredibly important for the construction of the appropriate glycans on the cell surface our within the cell. and i think it's also key to note that what john lions will talk about in a minute is pgm3, actually within this pathway for the production of udpglcnac.
so we have the sugar converted to the sugar nucleotide. now that needs to be in the right place at the right time. so the sugar nucleotides are by and large synthesized, the only exception, is acid sin theyised in the nucleiious. these suggests sugars can be
used by an enzyme that i'm going to talk about in a moment called ogt, but otherwise, these sugars need to make it into the lumpman of the er or gold g. and as you're well aware, these cellular membranes, including the membranes of the gold g and or biological barriers.
they're securityively permeable to a particular subset of moleculars. and because of that, and because ndp sugars are negatively charg ed, the cellular membrane is not permissive to the nucleotide that phosphate sugars there has to be a transport
mechanism. and there are a variety of transporters that are localized within the er and gogi that are responsible from the sight sole to the lumen of these organelles these are actually considered anti-porters, because they require the nucleoside mono
phosphate to be eflexed at the same time as the n ndp sugar is imported. you can see from this particular slide that there are a whole host of different nucleotide sugar transporters, one for each type of nucleotide. some of them are exclusive to
mammals, others present in multiple other organisms. and beyond the fact that there is one nucleotide sugar transporter or multiple for each individual sugar nucleotide, what's interesting, the fact that they're selectively localized.
you can see the acid, for example, that transporter is only localized within the golgi, because it's only utilized within the golgi. it has no role in the er. and like the previous -- one of the previous slides where i show ed you that there are
mutations in a number of these enzymes associated with disease, you can see that there are a number of these transporters that are also associated with and so cmp, sialic acid and gdp fucose are examples of enzymes that are in gly solelation, supporting the fact that the
synthesis of these nucleotide sugars is critical for the appropriate production of glycan now, like i mentioned before, i wanted to focus a little bit on udpglcnac. this are three transporters responsible for the correct localization of udp-glcnac.
it's present in a number of different compartments within the cell so it's made in the cytosol, and then it is imported into the golgi and er where it's concentrated. and the golgi and er, as john showed you, a number of those n link struct muched contain glcna
c. however, what i'd like to focus on is the use of udp glcnac within the cytosol. we're going from glucose here to the production of udp-grcnac. while it can be used for the gly solelation for a variety of these different types of protein
s, what i think is important to note is that it is critical for gly solelation of the simplist glycan. so glue seemmine hooked to a serine residue. one enzyme is responsible in addition to the target of proteins, so donor sugar,
nucleotide, ogt uses that donor sugar, and udp is the byproduct. and what i'd like to focus on, on this particular slide, is that the imports for the bio synthetic pathway are a different of different nutrients so they range -- requires glucose, glutmine
[indiscernible] and atp, which supports the fact that we think of o-glcnac as a nutrient sensor it senses changes within the cells ranging from nutrients to stress. include, o-glcnac is critically important for a variety of different biological processes.
it's important for regulating whether or not proteins are de graded. it's critical for whether proteins are folded or aggregate that be seen in a variety of neuro degenerative cities, for example, t tao is modified. whether a particular prepare is
o-glcnac will change the location, which may change the transcription of down stream gene one of the things we have been happening is when 0-glcnac is in disease? and if 0-glcnac is regulated appropriately, you have
physiological homeostasis. if it's perturbed you may have a disease state that begins and can end if some sort of nutrient change is ameluaterated or a stressor is removed. however, eventually, you may have a pathology that cannot change.
that can be seen if you remove o gt from mice, for example, that results in embryonic -- or in lethality. and so what i'd like to conclude with, and turn over the podium to john, is simply that 0-glcnac , but it is a nutrient sensor and down stream of udp
glcnac, which critical in you cleo tide sugar, important for not only o-gly knack but the pro duction of other glycans, is going to be essential for us to understand a variety of different types of diseases. again, i'd like to point out pgm 3 is part of this pathway por
the synthesis of udp-glcnac. thank you. [applause] the question was about sugars that can be modified biphosphate groups. so sugars can be modified in a number of ways including [indiscernible] as well.
those are found in those large- like -- the heparin -- not heparin. the -- i'll let you finish. >> so the sulfated use the paps donor, which is typically of all-- they're mostly in glyco protein hormones. and other rarer peptide hormones with regard to the phosphate,
the manos recognition system which holds enzymes in the er is mediated by a two step process. you transfer a glucosamine on to a terminal sugar and remove the capping glcnac so you end up with a terminal phosphate. that's actually a very, very a bundant phosphorylated species.
it's a good question. and actually, it was the basis for understanding the diseases. >> so john, is glycosylation a limiting factor in cellular re generation wound repair, cell division? >> very good question. michelle talked a little bit
about what's limiting. particularly with syntheses. how you're ontic acid is actual ly very important for wound healing, it's critical for wound healing. how it's the single biggest utilizer of udp glcnac, and it can limit the ability of the
pathway michelle just talked about, o-glcnac, from functioning. wound healing is actually limit ed by a formation of acid, it's one of the most abundant moleculars made. because it's one of the ur ontic-- it requires a trempool of udp glcnac.
we could have a demiss fid medicine just on this. >> do we have any others that want to ask questions? >> if not we'll have a break at the end. john can get up on here and answer the things we don't. five minute break?
>> i think -- >> bathroom break. >> all right. perfect. we'll get people out of here a little earlier. >> absolutely. >> i was thinking about an an analogy in terms of the way i
think about gly solelation. water came to mind. so this is nearly dried up reservoir outside of california, as you know, they have been in drought until just this winter. and when you think about gly sole layings, and water, both are entirely essential for life.
there safe garcia in place to -- safeguards in place to preserve water. as we learned in flip, michigan, if those safeguards go away, we end up with human disease. and finally, both with sugars and with water, it's likely just a little bit is not enough.
we know for water and we certainly think for for sugars as well, that we really need many of these to be abundant for life to thrive and for us to successfully do all the natural processes that are required for us to live as humans. so with that in mind, we'll move
on to some glycosylation dis orders. as a physician, i really focus primarily on the allergy side of things. in our lab, josh is my section chief as the name denotes, genetics and pathogenesis of allergy section, we've been
interested in single gene dis orders that lead to bad allergic problems. also look at known single gene disorders that have allergic problems to understand the mechanisms that drive the fundamental sort of predis position to allergy, as
well as severe allergic reaction s, such that we can apply them back to healthy people who have mild diseases. so these are a sort list of a dozen of show congentive dis orders, associated with immune dysfunction. you can see the majority of
these have been associated with something called nutropinia, or neutrophil chemo taxic defects. it may just be we notice these defects more. if you don't have neutrophils, you can get very, very sick. as shown in this micrograph, you can see the neutrophil here and
this is actually in a bladder where there is ecoli. this grand negative rods can cause inefficiency, like likely why we identified this in association with neutrophil defects. it's not all a problem of losing in some cases with
[indiscernible] which dr. hanover mentioned earlier. there has been a very interest ing story. this trimming enzyme which norm normally sort of cure curates complex and gallons in a particular way, when it's missing.
you have resistance to enveloped glycoprotein viral infection. this is a picture of a lymphocyte infected with hiv viruses. individuals with this disorder have resistance to this exact infection, as well as other glycoprotein enveloped viruses.
it's not necessarily the trade off individuals might be looking for it's certainly informative in biology and something we can learn from. so despite the number of des orders that are associated with immune changes, i'm really going to focus on pgm3 in part
because it's near and dear to me we worked in collaboration with a large group of folks, able to sort out what was going on. in the two families i'll discuss also it's incredible informative about how missing a early building block early in this process can have drastic
clinical effects that we're trying to get our heads around in terms of how these are going awry and how this is causing problems within these individuals. so i'll start with a story about a young boy who was sent here from egypt, referred for hyper.
ge syndrome. before i develop deep into that, i think we have to do a couple segues. the first is what is ige? you can close your eyes and nod back if you're aware, but if those who know less, this is the basic schema of allergic
sensitization and reactions, ige is the allergic antibody. this works in the right host, under the right circumstances, antigen or allergen is taken up by an antigen and presented to a t cell on class 2mhc in the form of peptide sitting on that class 2 where the t cell receptor
recognizes it, and as i mention ed, this has to be activated in the right sort of milieu, if you will, to drive this to bomb a thc cell ory learning effecter cell. and those th2 cells specific to that antigen make alearningic cytokines, il4, which promote b
cells also recognize that same antigen to make ige, which, then , gets into the blood, floats around, masks cells in the tissue. these are tissue allergic cells, born in the bone marrow, pre crewsers, and float until they deposit in your skin, nose,
places you may have any learning ic reaction. they reach into the blood and grab ige with these long tend dals, on these high affinity receptors. so then you have had this whole priming event happen. let's say it's peanut.
the next time you see peanut, that protein can cross link this ige and cause massive release of these mediators of acute and chronic allergic inflammation, such as history tomean. which is why antihistamines pre vent hay fever. this can be short lived or whole
sail. so there is varying degrees of why this happens, lots of detail s that i'm happy to discuss at the end, but it's beyond the copy scope for the that's ige. there are individuals classified having hyper ige.
we don't like this kind of label because we think it can be a little bit misleading, if it's interpreted tin wrong -- or mis interpreted in the wrong context. so i'll kind of give an example of a few disorders that cause high ige, the vast majority of
people do not have a known dis order. so this young boy, 12 years old, was reared from equip with a hyper ige syndrome. he had significant elevation in ige, 300 times the normal, about 30,000. international units.
he had moderate to severe atopic dermatitis, itchy skin, food allergies to multiple foods, asthma, allergic rhinenitis. also had some infections. recurrent skin infections. he was having recurrent pneumonias, and chronic draining , external ear infection
s that chronically drained on him. what was also interesting about this man, he had very low white blood cells, low nut nut -- neutrophils, also low lymphocyte so our second sega, then we've continue to move on. to give you a sense of what this
is, at least a couple of conditions that can lead to hyper ige in terms of genetic mutations. there are two forms that have been described in the literature up until now, and some might consider pgm3 among them. there is autosomal recessive
forms, loss of mutations. doc 8, it's an important molecule for regulating acton filament movement, it's an ex change factor but the role is thought to be related to cyto skeleton rearrangement. these individuals have sort of a number of traits, but across
them and individuals with the dominant form, the first described form of hyper. ge syndrome caused by these negative mutations, actually, the try ad of high ige, and skin or lung infections was the initial description. and both of these do have those
things but importantly, what we see in doc 8, it has somewhat of a combined pro segive immuno deficiency. this is less the case in stat 3, certainly they have problems with t cell mediated memory and with help moral or antibody pro duction, but not as severe as
the dock 8 patients. we also see a heck of a rot more severe allergy in dock 8 patients than stat 3 patients. further, i'll show you a picture in a second. cutaneous viral infections. really fro profound. negatively impacting the lives
of patients. in dominant negative stat 3 patients these are much less prominent and less dill tar i can't say. and finally, there is a whole connective tissue story we see in patients with stat 3 dominant negative mutations.
i should mentioned this, this is the job -- job syndromes. they have scoliosis, a curbed spine or they hand on to their baby teeth. so they have many teeth that have to be extracted which we don't see in dock 8. i include here severe determine
tighting. they can have higher ige than patients with dock 8. certainly some of these are probably genetic diseases, mono genetic disorders. but certainly environment and components contribute. and many of these other features
are not seen. so lastly, i should mention that for the purposes of today's talk , there is a concept of th17 mediated immunity. we don't know a ton about it. it's been studied a lot but i think there is still dis agreement as to what it's
doing. we certainly think it has a role in fungal immunity in terms of protecting us for certain kinds of yeast and fungal infections. it's associated with a number of autoimmune disorders, particular ly multiple sclerosis and cirrhosis among others.
but job's patients certainly don't have th17 cells to speak of. that's important to contrast to what i'm going to share with you about p-gp 3. that's the back crop. i guess the last point -- sorry, i get droning on.
nudgerlogic defects are not present in these folks either. so here is a couple of pictures of patients with high ige states here is a patient, these are quite significant waters on every finger, makes it miserable to deal with. here is a patient with severe
curvature of the spine, cervical instability due to stat 3 but here is an individual certainly does not have normal skin, this is severe atopic dermatitis, where it's quite inflamed. and this individual has no known mutations, certainly has an ige
in the 50 to 60,000 range. certainly might get lumped into this hyper i ige syndrome. so getting back to our patient. importantly, this individual was born from a -- his childhood was fairly uncomplicated. the pregnancy was uncomplicated, no infections during his 9
months. there were no complications at births. he was worn by c section. and earn childhood, no illnesses but shortly after a couple of years of life he started to develop recurrent infections of the skin, the lungs, ended up getting hospitalized in egypt.
i apologize for the quality of this image. we were ecstatic to get one. this is reconstruction and this is a ct scan, not fantastic quality but sufficient to real ize that one of these things is not like the other. we don't have to be radiologists
to rile ease there are multi focal infiltrates, you can see at the right lung ways, not present in gross inspection in the left lung but there are other multi focal densities on that lung as well. so this is a must focal pneumonia, the real deal which
can be quite tricky to tease out in a kid. we were excited to have hard evidence that this was an infection. we don't know whether this was bacterial. it may have been bacterial and fungal.
he was given broad spectrum and iic ain't biotics. he did not have any neurological deficits but we did notice there were characteristic neurological differences in this child, and we wanted to understand what was going on and how to characterize them.
he went under physiatric testing , had clays development in terms of reasoning level at age 11, when he had that evaluation. he was reasoning at what was determined to be roughly 6-year old level. and he was having quite a bit of
difficulty visual spatial problems. so neuro psychiatric testing, a series of different tasks to assess what skills and abilities and understanding and emotional responses you have to a number of different stimuli and questions.
he was -- he displayed clinical ical discord nation, im paired motor control and visual tracking. he had a little bit of speech difficulty where he would sort of have delayed speech and discord nation and slow processing.
and he had a history of febrile seizures. we don't know whether that was related. so we wanted to get a better sense of what kind of functional change we were dealing with. and so i was trying to get a good picture of what he under
went. i thought maybe we would just try it. this is something similar to what we would call a visual evoked potential. i hope if anybody gets motion sick, close your eyes for a second.
i'll tell you when it's over. basically, a patient is asked to zaire at a screen. there are boxes that are black and white. they have probes attached in the back of their kid. the screen kind of forecloses. when that happens, this is
actually a real fantastic functional mri picture of someone undergoing this clinical experiment. you can see this sort of tracks of the -- the eyes would be up here, these are the optic tracks , the radiations. this is the visual cortex that
you can see light up. what we can do is by putting probes back here, just on the outside of the head and passive ly flashing lights we can determine whether he has changes in neurologic conduction. we don't have to stick needles in him or do anything painful.
so i'm no neurologist. what i can tell you, when this happens, you have the evoked event, this trashing light. and then you have this change and the second dip is what they measure, p100 value. in a normal individual, you can see that each of these boxes is
30 million seconds. then you can see that each box this way is ten micro volts. normally, it takes 90 seconds to get to this dip. in our patient it took about twice as long. so every time he's seeing something it takes twice as long
to get to the visual cortex as it would a normal individual. this corresponded nicely to some interpretation of some fancy mri images that danny, one of our collaborators, wasabling to help w what you can see is that these very subtle light areas, where they should be dark.
and this is hypomyelnation or dismyelnation. we're not certain. doesn't look to be progressive, fairly stable other time but it corresponds nicely to this delay ed signal propagation. and just to remind you, myelin is these insulators that allows
for rapid propagation of neurologic synapse information. so importantly, interestingly in our patients, this kind of dis myelination pattern looks a lot like multiple sclerosis. these patients do not have multiple sclerosis but interest ingly, a collaborator of
ours at uc irvine has been able to demonstrate a number of mouse models and as well in humans, that in fact environmental genetic changes in certain pathways that alter glycosylation seems to predis pose patients to multiple sclerosis, similar demyelin
nation pattern. in mouse molds, it seems this can inhibit, in particular, the th17 group i was talking about a little bit ago. which as i mentioned was associated with things like psoriasis. this man had a lesion that
looked like this in which there was a plaque that was well demarcated, scaling on the surface with a dark base, which was consistent with a plaque, looks like plaque psoriasis. consistently, i don't share -- these individuals have quite a few th17 cells relative to
controls. so it all fit together. so in addition to the index patient i mentioned we were able to have -- speak and meet meet the worry and a cousin who came to nih for evaluation over a course of a year-and-a-half who had a lot of the same problems.
the younger brother of our patient had a lot of allergy problems, skin infections, neurologic problems, auto destroying his red blood cells. we're not sure if that's an autoimmune problem. had significant elevation in. ge, normal is less than 90.
that's quite high. as well as other high immunoglob leaderships, low lymphocytes. the 9 month old cousin who's. ge level was not terribly had, had significant dermatitis. 9 months was unable to sit un assisted, not a typical milestone.
quite delayed. and neutropenia. so whale we were working with alex freeman and folks to evaluate these patients, work up what he was going on and per forming sequencing, another family here had been followed for a decade and a half, maybe,
that jennifer puck, when she was here, working with steve holland-- i think before the lc id was the lcid. followed a family and described them in which they identified immune 0 deficiency, they had 5 effected children and it's non consegious june union, very bad luck.
[indiscernible] should mention the vasculitis is a similar autoimmune process, there is the neurologic problem. what about the aledger? this is a clinical photographer of one of the effected i hope you can appreciate the severe nature of his dermatitis
in an area he can't scratch. there was skoallosis present in his family. when you look back through, virt ually all those kinds of findings we were seeing in our index family were found in this family. and through sort of a
collaborative coincidence, another group was sequencing this family. helen haves lab was -- who discovered dock 8 sufficiency was working on these folks. we came to the gene at the same time. that was pgm3 in both these
families. so our family that we initially sequenced was homozygous for this d325e mutation. whereas the other family had this frame shift and another -- mutation in the phosphate bind ing domain, and frame shift prepare was not expressed on the
transcript level. you can see this -- in this model of the enzyme -- we should have said, mutase 3. has two mutations. the two mutations. the phosphate is shown here. blown up here. you can see they sit right sort
of at this -- at this active site where glucose 1 and 6 phosphate are inner converted. so we think what's going on is that those mutations not allowing it to get into that space to actually have that transfer happen of that phosphate.
and leading to a problem. so i'm going to elaborate on the allergy, to remind you, allergy is part of this picture. infections are part of this picture. autoimmune, we think s part of this picture and neuro cognitive impairment.
this was a new syndrome, a new constellation of traits associated with this pgm3 yoman's work began of trying to figure out how this happened, how these diverse problems were arising from loss of the single i should mention since our characterization of these 2
families, our collective characterization, there is now 29 individuals from 13 countries described across the world with this disorder. the clinical phenotypes are even broader, including skeletal dysplasia in a number of i'll show you the first report
of severe immunosufficientcy here, but there have been subsequent reports of skid as well in other pgm3 deficientant many of these patients have been successfully transplanted and doing quite well. so what's at the crux of this? i think michelle set this up, so
great to go after these guys. the fundamental glycobiological has been described better than i could do it. as mentioned, pgm3 is very important for the production of that udp, and working with the hanover lab we were able to show that udp-glcnac levels are low,
both in peripheral blood but also in primary dermal fibroblasts. we looked in a couple different compartments. so the reason i got so interest ed and the reason i think this is so fundamentally important for our understanding
ofy learningic diseases, that these individuals have every form of allergic disease. talking about 7 people. who have essentially every form of allergic disease that i know of, except for contact dermatitis. which is sort of an atypical
kind of allergying problem. and so it seems that udp glcnac is particularly important for the development of a myriad form s of allergic disorders. if we can understand the fundamental mechanism, perhaps we can learn quite a bit of the under lying pathophysiology.
so, as i mentioned, udp is fundamental to n glycans. we wanted to look, one, could woe find abnoral glycosylation on these patients and could we screen better? because thee guys -- some of these guys may have been missed with traditional screening
techniques. and so in addition to that core, butting core n glycans that john describe earlier, inside of the er and golgi, udb glcnac is incredible important for serving branching. one form is here, entirely dependent on it.
mike has done a lot of work in this, where he's looked in both ms patients and mouse models showing that this particular arrangement of udp glcnac, particular blanching arrangement is entirely dependent on glcnac levels. if the levels were true and
lower in our patients we should see reduced branching of this kind of complexity of n glycans. there a lectin, that you can get from red kidney beans that recognizes that. you can purchase it as a flora conjugate and that's what we did. we have been able to establish
an assay where we can stain just peripheral blood, mononuclear cells with a number of markers and look in different the defects seem to be compartments willed. i won't go into that today but when we look in naive cells, i'm showing you cd4, but also cd8 as
well, we some substantial re ductions using this lpha leken based flow cytometry assay. when caring to other disorders that we described earlier, of hyper ige, as well looking at controls. this is a nice screening test because the high est value we obtained from p
gm3 doesn't joe lap the 99% confidenceiority val of the low est of the group. we have started to use this clinically for other patients with pgm3 deficiency or similar glycosylation disorders. hadn't found any yet but we're looking along with folks in nhgi
how might n glycosylation effect the kind of immunologic and allergic free throws? and we have some -- phenotypes. we have some data. i'm going to share the schematic i have the data if people want to look at it. there is a molecule on t cells
called ctla-4. this is a break on on the t cell following activation. this is upregulated, expressed on the surface that a normally co stimulatory molecule sort of binds to it and says no, don't go further, calm down. when that happens, the immune
system gets a red lighted and the t cell activation is sort of held in check. well, it turns out that ctla surface expression is entirely dependent on appropriate glycosylation of that molecule. it's not a problem of making it. even if it's there, the the
glycosylation is wrong, it doesn't say on the surface. when that happens, a different molecule that's already on the surface can kind of jump in this , cosometimely model called cd28. that can be recognized, recognize that same ligand, if
you will, which gives the t cell a green light to proliferate and become an activated effecter. when that happens, t cell proliferation is under strain. we see inflammation. through a process that -- has been sort of the life's work of michael lab, this whole idea of
activation induced cell death where this unrestrained act ivation leads to actual -- eventual death of the t lymphocytes. so we can explain both potential ly, at least in one mechanism, both the lymphopenia as well as the unrestrained
inflammation using this model. we're working to confirm that ct la is glycosylation. so in addition to altered gly can complexity and potential effects, and obviously, the vast majority of immune molecules are gly soleleted. it's not limited to that.
i'm going to speak about how this might contribute to allerg ic problems. again, this is very -- it's in its early days of vetting but 0- glcnacation -- it's really important, what makes it very complicated and difficult to study, is that a number of
remarkably important transcription factors and pathways are effected by this process. and virtually all of them can be effected in 0-glcnacuated. so our sort of typical technique s of understanding how these pathways are activated,
for instance, stat 3 is phosphorylateed. glcnacilous may effect -- phosphorylated activity without affecting whether it's in the thus or phosphorylated. so we set out to look to see whether we could see abnormalit ies in pg3.
we saw they had reduced levels. we should see less 0-glcnacation we look and we didn't see a difference. we are scratching our heads. what the heck? sh should be fixed. as it turns out, typically, biological is more clever than
we are. and so what these patients cells had done is up regulate ogt in significant excessive controls. and downregulated the enzyme that remains that glcnac oga, such that they can maintain at least in mass or a large scale level, normal 0-glik nation
layings of the proteins within the cells at the exsense of almost no expression or activity so why is that important? well, john hanover's group has made the oga knockout mice. we were able to mine some of the data and done our own mass spec experiments to try to evaluate
this further. what we can see is that the transcriptional profile in oga deficientant mouse embryonic fibroblasts basically have a similar predisposition, as we might predict in our patients. we see up regulation of genes involved in alearning priming
and th2 differentiation, as well as information, and down regulation of th1, th17 -- immunomodulatetry modules, as well as other stop gaps that normally suppress the kind of inflammation we're seeing in the patients. and so what we think is going on
is that it's known that the rapid on off of these 0-glcnac residues is quite important for a number of cellular processes. in these folks with the lack of aga, we don't think these are -- once it's on, it's stuck on long we're working to prove that right now.
so -- but kind of the last bit of the talk is just that in any of these kinds of situations we're always looking for a way to treat or modify the disease. and certainly, in these folks who have been refractory to many kinds of therapy, we're looking for a new way to approach this.
so we were able working -- this is data we worked on with have you had freeze's lab at the in stitute, we were able to show that exogenous glucose mean in fibroblasts -- these primary fibroblasts, was able to rescue the phenotype. shown in blue, more udp quick
knack at baseline, but the addition of udp glcnac was able to restore the total levels of u dp glcnac in the fibroblast of p pgm3 in purple, to no difference from controls in red. and more recently, looking at these oga and defects we were able to show again that the oga
activity was subject substantial ly lower at baseline. with increasing amounts of glycosylation we could normalize the oga activity in these folks. so i guess it was about a year-and-a-half ago now, based on these findings, we initiated a protocol to treat these
patients or subject these patients, i guess, to supplement ation to look at any structural and functional change s in the immune system. i didn't have time to get into it here today, but certainly what we've seen is that rather than -- wheel wake restore the
-- we can restore what looks to be the sugar levels and glycan problems in these patients, peripherally restored the effecter cells aopposed to the regulatory cells. we think we have a handle as to why that is, a whole new hurdle to overcome.
we hope base on these exempts we continue to develop new therapies, not just for pgm3 but also for people with allergies in general if this tends to be a common theme. as with many clinical studies and projects, there is a large and vast number of collaborators
eboth in basic sciences as well as in the clinical side. i just have to mention alex and derk who helped -- alex is sort of the staff physician who is -- who has done the vast majority of the clinical care of these derk is their study coordinator.
the lab and michelle has contributed quite a bit to this work, as john mentioned at the outset, i do continue to work with folks in the [indiscernible] particular, hngr i looking for more of these desorders. and with that i guess we can
answer more questions' people have them. thanks. >> well, thank you. [applause] do we have any questions? if you do, please speak up. >> you mentioned the [indiscernible]
>> complete deficiency, yes. >> complete deficiency. going back to your mutations that are defective in the frame ship region in the binding domain, i believe it's almost -- >> no, that's compound. >> ah. >> yeah.
so -- >> that mutation [indiscernible] >> so we're making a conditional knock in mouse right now of the other allele. >> frame shift. >> the frame shift is a stop gain. it's not expressed.
so it's compound with this [indiscernible], and then the other patients are homozygous for this d325. >> so the second question, there are high km andlo km enzymes. the virus edition probably -- the first edition, probably the lo km.
further, maybe they require high er concentration. so how does it feed into the [indiscernible]? is it the levels? so [indiscernible] >> draft hanover is probably the best person to explain that. my impression of it is it's the
udp -- the udp glcnac level sensed through the mechanism of 0-glcnac. the concept is similar to that of hypoglycemia, in this setting so at least that's how i think about it where these patients essentially to the cell, are walking around as if they're
hypoglycemic. and that is leading to this auto regulation of those two enzymes to try to maintain a normal glcnac level. >> the one thing that -- for those who are interested in early embryonic development, these enzymes are associated
with the hawk's gene clusters, principally involved in the whole formation of the met azon en body plan. they're actually in the hux clusters that are regulated by high order chromatin structures. this pathway evolved, is really a genomic responder to nutrients
so kind of gives shivers down my back when i think about that. it really is probably key to understanding a lot of these met abolic disorders associated with transcriptional regulation. >> and it happens remarkably rapidly. so if we take primary t cells
from patients, and just simply withdraw glucose and glut mean from the culture medium, within an hour we see massive down regulation. and within a few hours we see changes in gene transcription. >> this may be wild but people with people with deficiency
respond to immuneization with glycan linked -- >> not wild at all. so it's a complicated question. because their responses are not normal. they're deregulated anyway. as far as -- in as far as we can tell, they can respond to
[indiscernible] fairly well. poly sacried, not well but well enough we think to be mostry protected. one of the individuals here actually had a [indiscernible] nephrites that required [indiscernible]. but as you can see one of the
individuals total was 2,000. some of that was specific to immunizations. >> now, functionally, does it work as well if the n glycosylation is different. so far as we can tell, plasma cells, we think because of met abolic changes and changes in
gene expression ra relatively normal in these patients. when people looked a immuno globulins -- there is a great story about altered fc and glycan expression or changes in the n glycan of the fc on ige, drives anaphylaxis, or can completely obliterate any risk
for apflaxis. when people look at poth, they have not seen significant differences in n glycan expression. >> well, i want to thank you, thank you all. that was very exciting, very complex world.
but these -- all the power points are on our website. and accessible, as well as selected readings, so people can read further and think about this. thank you very much. it was really very exciting.
