Golgi compartments enable controlled biomolecular assembly using promiscuous enzymes
Abstract
The synthesis of eukaryotic glycans – branched sugar oligomers attached to cellsurface proteins and lipids – is organized like a factory assembly line. Specific enzymes within successive compartments of the Golgi apparatus determine where new monomer building blocks are linked to the growing oligomer. These enzymes act promiscuously and stochastically, causing microheterogeneity (moleculetomolecule variability) in the final oligomer products. However, this variability is tightly controlled: a given eukaryotic protein type is typically associated with a narrow, specific glycan oligomer profile. Here, we use ideas from the mathematical theory of selfassembly to enumerate the enzymatic causes of oligomer variability and show how to eliminate each cause. We rigorously demonstrate that cells can specifically synthesize a larger repertoire of glycan oligomers by partitioning promiscuous enzymes across multiple Golgi compartments. This places limits on biomolecular assembly: glycan microheterogeneity becomes unavoidable when the number of compartments is limited, or enzymes are excessively promiscuous.
Introduction
The surfaces of all living cells are decorated with informationrich oligosaccharide molecules known as glycans: branched sugar oligomers covalently linked to proteins or lipids (Varki et al., 2017). Glycans encode cell identity and mediate a variety of intercellular interactions. They play critical roles in development, species recognition, selfnonself discrimination, and hostpathogen coevolution (Varki, 2017).
Glycans are composed of a small set of monosaccharide building block types (monomers) and disaccharide bond types (linkages in the branched glycan oligomer) (Adibekian et al., 2011; Figure 1A). Eukaryotic glycans are built by collections of glycosyltransferase (GTase) enzymes the ER and Golgi apparatus, a process known as glycosylation. GTase enzymes are chemically precise but contextually sloppy (Taniguchi and Honke, 2014; Biswas and Thattai, 2020): a given enzyme catalyzes a specific bond between a specific pair of monomer types, but can act promiscuously and stochastically on many oligomer types (Figure 1B). As a consequence, even a single cell with a limited set of GTase enzymes can theoretically synthesize an astronomical array of oligomeric combinations (Cummings, 2009).
A given glycosylated protein type in a given cell is usually associated with multiple glycan oligomers, a form of moleculetomolecule variability known as microheterogeneity. The enormous potential for variability is exemplified by prokaryotic glycans, which are typically random heteropolymers (Varki et al., 2017, Chapter 21,22; Adibekian et al., 2011). In contrast, eukaryotic glycoproteins are typically associated with a narrow, specific set of glycan oligomers, referred to as protein’s glycan profile (Varki et al., 2017, Chapter 1; Campbell et al., 2014). The low variability of eukaryotic glycans is functionally relevant: specific protein glycan profiles are associated with distinct species (Hamako et al., 1993; Hård et al., 1992; Hokke et al., 1994), distinct individuals (as with ABO blood groups [Patenaude et al., 2002]) and distinct cell types in an individual [West et al., 2010]; and altered glycan profiles are implicated a variety of disorders (Freeze and Ng, 2011; Gill et al., 2013; LoGuidice et al., 1994).
How do eukaryotic cells generate narrow, specific protein glycan profiles despite the variability caused by promiscuous enzymes? Simulations suggest that the localization of enzymes within Golgi compartments plays a key role (Liu et al., 2008; Spahn and Lewis, 2014; Spahn et al., 2016; Fisher et al., 2019). Consider the GTases Fut2 and GTB, which specifically synthesize the blood group Bantigen (Taniguchi and Honke, 2014, Chapter 44,47). Hypothetical promiscuous versions of these enzymes, if placed in a single compartment, generate variable tandem repeat oligomers as byproducts (Figure 1C); specific synthesis of the Bantigen is restored when the promiscuous enzymes are placed in two separate compartments. However, tandem repeat synthesis is just one among many sources of variability, and GTases can be more or less promiscuous than the enzymes in our example. We would like to understand the general conditions under which compartmentalization mitigates glycan variability.
Mechanisms for controlling variability are central to the field of algorithmic selfassembly, which explores how building blocks with sloppy interactions can be programmed to assemble into a desired final structure (Soloveichik and Winfree, 2007; Zeravcic and Brenner, 2014; Murugan et al., 2015). Here, we use ideas from selfassembly theory to precisely enumerate the possible sources of glycan variability. Using rigorous mathematical theorems, we demonstrate that compartmentalization mitigates variability for large classes of promiscuous enzymes (Figure 1D) (proofs of theorems are provided in Appendix 2). Since the number of distinct Golgi compartments in any cell is limited, microheterogeneity due to byproduct synthesis becomes unavoidable during the synthesis of complex oligomers. Nevertheless, multicompartment synthesis greatly increases the set of oligomers eukaryotes can specifically synthesize (Figure 1D), complementing other mechanisms of enzymatic control such as kinetic regulation. Thus, a quintessential eukaryotic trait (intracellular compartments) provides the means to overcome a fundamental biochemical limitation (enzyme promiscuity). This capability may underlie the conservation of the Golgi across eukaryotes (Barlow et al., 2018): the idea of the Golgi apparatus as a factory assembly line is more than a metaphor, it is a mathematical and biological necessity.
Results
Glycan synthesis in Golgi compartments
We focus on the diverse class of Oglycans, which are associated with most eukaryotic cellsurface proteins (Varki et al., 2017, Chapter 10). The synthesis of Oglycan oligomers begins in the Golgi apparatus, when a root monomer is attached to a specific serine or threonine on a substrate protein. The Golgi consists of an ordered series of compositionally distinct compartments (for example, cis, medial and trans cisternae). Each compartment contains a specific set of GTase enzymes responsible for growing an oligomer on a specific protein type. Multiple models are proposed for how oligomers and enzymes are trafficked through the Golgi. In the transport model, oligomers are ferried through successive compartments; in the maturation model, oligomers remain in place while the enzymatic composition of the compartment undergoes a rapid switchlike transition between successive states (Mani and Thattai, 2016; Pantazopoulou and Glick, 2019). Under either scenario, whether transport or maturation, growing oligomers spend time within successive enzymatic compartment types. (The residence time of an oligomer within a compartment type is exponentially distributed in transport model, and sharply peaked in the maturation model; our results only depend on whether the average residence time $T$ is small or large compared to the average time for monomer addition.) The oligomer finally exits the last compartment in the series. The set of final oligomers associated with a given substrate protein makes up its glycan profile.
GTase promiscuity and stochasticity
As the growing oligomer spends time within successive Golgi compartments, it encounters distinct collections of GTase enzymes (Moremen et al., 2012; Pantazopoulou and Glick, 2019). During each such encounter, the enzyme scans the oligomer for a site that matches a structural motif (the ‘acceptorsubstrate’) and attaches a single free monomer (the ‘donorsubstrate’) to that site (Figure 1A; Liu et al., 2008; Moremen et al., 2012). A given GTase enzyme invariably catalyzes a glycosidic (COC) linkage between specific carbons on a specific free donor monomer type and a specific acceptor monomer type within the acceptorsubstrate (Figure 1A). However, these enzymes are doubly sloppy: they are both promiscuous and stochastic.
Promiscuity
There are two strong reasons to expect GTase enzyme promiscuity. First, there are far fewer GTase enzymes than observed glycan oligomers in any given species (Narimatsu et al., 2017); moreover, the same highly conserved enzymes are found across species with very distinct glycans (Kaneko et al., 2001; Figure 2). Therefore, by the pigeonhole principle, an individual GTase enzyme must be promiscuous and able to act on many distinct oligomer types (Narimatsu et al., 2017). Second, although oligomers can be arbitrarily large, there is a limit to the size of the motif any enzyme can recognize within it. A given enzyme could act everywhere its recognition motif is found on an oligomer; the smaller the recognition motif, the more oligomers it will be found within, and the more promiscuous the enzyme (Blixt et al., 2008; Taniguchi and Honke, 2014; Biswas and Thattai, 2020). In our analysis we consider three broad classes of enzymes, exemplifying varying degrees of promiscuity (Figure 1B). Contextfree enzymes are those whose acceptorsubstrates are single monomers, no matter what branches or roots they are linked to. These enzymes are maximally promiscuous. Ideal branchsensitive enzymes are those whose acceptorsubstrates are some acceptor monomer type having or lacking specific branches. These are intermediate in promiscuity. Ideal rootsensitive enzymes are those whose acceptorsubstrates are some acceptor monomer type linked to a specific root chain. These enzymes can distinguish every monomer in an oligomer and are minimally promiscuous. Enzymes that read roots and branches to partial depth represent more complex types of intermediate promiscuity; we do not discuss these possibilities here.
Stochasticity
Chemical reactions within cells are necessarily stochastic, due to the low molecule numbers involved. While the growing oligomer is within a given compartment, the order in which it encounters the available GTase enzymes is equivalent to a Markov process of random sampling with replacement, with randomly distributed time intervals between successive encounters (Gillespie, 1977). The reaction network of the compartment shows every possible oligomer growth order starting from a given input oligomer, as a result of all possible enzymecatalyzed singlemonomeraddition reactions in all possible permutations (Figure 2A). Since Oglycan oligomers are not pruned, these reactions are irreversible (Varki et al., 2017, Chapter 6). Within a reaction network, intermediate oligomers are those that can potentially be further extended by some available GTase enzyme, and terminal oligomers are those that cannot be further extended. Two identical input oligomers might take different paths in the reaction network as they encounter GTase enzymes in different random permutations and at different times. An oligomer might encounter the same enzyme repeatedly (if the enzyme is at high concentrations), or it might exit the reaction compartment without ever encountering some enzyme (if the enzyme is at low concentrations).
Patterns of glycan variability
Consider any assemblyline reaction in which oligomers are built by adding one monomer at a time. We can show that there are precisely three ways for variability to occur (Appendix 2, Remark 1): truncated, runaway, and divergent reactions. Each type of reaction variability corresponds to a specific output pattern that has been observed in real glycan datasets (Figure 2; Table 1; Varki et al., 2017, Chapter 1). These are exemplified by oligomer profiles of respiratory mucins from a cystic fibrosis patient (LoGuidice et al., 1994), human chorionic gonadotropin (CG) from a cell line (Hård et al., 1992), and horse chorionic gonadotropin (Hokke et al., 1994) (datasets from UniCarbKB [Campbell et al., 2014]). These patterns can be explained by the following enzymatic causes. For the mucins, two identical input oligomers exit the reaction compartment at different stages of growth; since some reactions are truncated, this gives a combination of both intermediate and terminal oligomers as outputs. For horse CG, a compartment contains two enzymes that drive a runaway reaction; this gives oligomers with an arbitrary number of tandem repeats. For both mucins and human CG, two enzymes compete for the same acceptorsubstrate; this sets up a divergent reaction, with mutually exclusive oligomer fates depending on the random order of enzyme action.
We model stochastic assemblyline reactions as continuoustime Markov processes with constant transition probabilities per unit time. This provides a probability distribution over each possible fate of the final output oligomer. The Shannon entropy of this output distribution in bits captures the variability of the glycan profile; approximately, it is the logbasetwo of the number of distinct highabundance oligomers. Each source of variability makes a distinct contribution to the residencetimedependent entropy, as seen for the hypothetical reaction networks in Figure 2. For horse CG, the input entropy of the final compartment is zero (since there is a unique input oligomer); for human CG and mucins the input entropy is 1.5 (since input oligomers are in a 1:1:2 ratio due to divergent reactions in earlier compartments). At short residence times, the entropy initially rises due to the exit of intermediate oligomers from truncated reactions. This corresponds to the mucin profile. At long residence times multiple intermediate oligomers converge to a few terminal oligomers, so the entropy decreases; this corresponds to the human CG profile. For the horse CG profile, the entropy stays high even at long residence times, due to synthesis of tandem repeats via runaway reactions.
Enzymatic causes of glycan variability
The examples discussed above suggest that each type of glycan variability is connected to a distinct enzymatic cause. We now make this connection rigorous (Table 1).
A truncated reaction causes intermediate oligomers to be produced as outputs. This occurs whenever the average waiting time for monomer addition (a quantity inversely proportional to enzyme concentrations) is comparable to or greater than the compartment’s residence time (Appendix 2, Remark 1). The only way all input oligomers are guaranteed to reach a terminal state (assuming no proofreading) is if enzyme concentrations are sufficiently high, or equivalently, the residence time $T$ is sufficiently long (schematically, $T\to \mathrm{\infty}$).
A runaway reaction is an infinite path in the reaction network, giving oligomers with arbitrary numbers of tandem repeats as outputs. This implies at least one enzyme must act repeatedly along a single chain. Ideal rootsensitive enzymes don’t permit runaway reactions, since their acceptor monomers are at a fixed depth from the root. To diagnose runaway reactions for branchsensitive and contextfree enzymes, we examine the compartment’s linkage network, summarizing the allowed order of monomer linkages (Figure 3A; Appendix 2, Remark 2). A compartment contains a runaway reaction if and only if its linkage network contains one or more loops (Appendix 2, Lemma 1), since each loop corresponds to a tandem repeat. Branchsensitive GTase enzymes can prevent linkage loops by using triggers (branched acceptorsubstrates that cannot be synthesized within the compartment from a single monomer; Figure 3D).
A divergent reaction is a fork in the reaction network that never reconverges, with distinct paths leading to mutually exclusive oligomer fates as outputs. To diagnose divergent reactions, we examine the acceptorsubstrates of every enzyme in the compartment. A fork occurs whenever distinct enzymes can act on the same oligomer to yield distinct products. If these enzymes could act in any order (for example, if they act on distinct empty acceptor monomers on the oligomer) then the reaction paths could reconverge after the fork. If a fork does not reconverge this implies that the action of one enzyme on an oligomer blocks the subsequent action of another (Appendix 2, Lemma 2). For rootsensitive and contextfree enzymes, this can only occur if both enzymes compete to act on the same carbon of the same monomer (bidirectional acceptor block; Figure 3B). For branchsensitive enzymes, there are two ways this can occur: both enzymes have the same acceptorsubstrate (bidirectional acceptor block; Figure 3B) or the acceptorsubstrate of the first enzyme is on a branch of the acceptorsubstrate of the second (unidirectional acceptor block; Figure 3B).
A compartment containing an arbitrary collection of enzymes might permit runaway and divergent reactions (Figure 3C, top). Our analysis shows that these sources of variability are not due to any individual enzyme: they arise from interactions within collections of enzymes (Figure 3A,B). We can eliminate truncated reactions by requiring high enzyme concentrations; we can eliminate linkage loops and acceptor blocks by removing certain enzymes from the reaction compartment (Figure 3C; Appendix 2, Lemma 3). These steps result in an ‘algorithmic compartment’: for each possible input, it specifically synthesizes a corresponding unique output. If we could watch an individual oligomer growing within an algorithmic compartment, we would find that its growth order always satisfied certain special properties that are easy to check, which we collectively characterize as ‘algorithmic growth’ (Appendix 2, Lemmas 4,5). The characteristics of algorithmic growth depend on the degree of enzyme promiscuity (Figure 3D). We now discuss how the concept of algorithmic growth can be used to find the minimum number of compartments required to specifically synthesize any desired oligomer.
Controlled glycan synthesis in multiple compartments
Suppose we want to specifically synthesize a given target oligomer from a given input oligomer, with no byproducts. As a first attempt, we might pick an arbitrary growth order that leads, one monomer at a time, from input to target. Each monomeraddition reaction corresponds to the action of some enzyme. We could simply load a single compartment with this set of enzymes. This guarantees that the target oligomer will be synthesized from the given input. The problem is, various byproducts might also be synthesized due truncated, runaway, or divergent reactions; and the target oligomer might itself be further extended at long residence times. Since these problems arise due to interactions within collections of enzymes, they might be avoided by splitting the enzymes across several compartments. The following theorems provide the answer to two questions (Figure 4A; Appendix 2): Is specific synthesis of the target from the given input even possible? If so, what is the minimum number of compartments required?
Theorem 1
A target oligomer can be specifically synthesized from an input oligomer if and only if it can be specifically synthesized from that input oligomer in a series of singleenzyme compartments.
Theorem 2
A target oligomer can be specifically synthesized from an input oligomer in a series of $N$ compartments if and only if there is a growth order from the input to the target that can be fully decomposed into $N$ algorithmic growth stretches.
Theorem 1 provides an efficient protocol to search for a solution: we needn’t consider all possible multienzyme combinations, it is sufficient to check singleenzyme compartments (Figure 4B). Once a solution is found using singleenzyme compartments, Theorem 2 provides a protocol to construct a solution using fewer multienzyme compartments. In this way, we are guaranteed to find the minimum number of compartments required for specific synthesis of the target (assuming a solution exists). For ideal rootsensitive enzymes a single compartment is always sufficient, since every growth order to a given terminal oligomer is algorithmic (Appendix 2, Lemma 4). The minimumcompartment path could be longer for the more promiscuous branchsensitive or contextfree enzymes (Figures 1D and 4B). There are some situations in which glycan variability is unavoidable: certain oligomers can never be synthesized without byproducts, no matter how many compartments we allow. In Appendix 3—figure 1 we discuss an example of a target oligomer that cannot be synthesized using branchsensitive enzymes in fewer than two compartments.
Discussion
Living systems excel at building complex structures using stochastic, unreliable molecular components. Macromolecules such as DNA, RNA and proteins are built by copying known target templates, so errors can be removed by proofreading (Murugan et al., 2012). However, most biological structures are encoded using a stepbystep recipe – that is, an algorithm – rather than a template (Navlakha and BarJoseph, 2011). This is apparent during animal development: the genome encodes a recipe to make an adult, it is not a homunculus of the adult. Eukaryotic glycan synthesis is an exquisite demonstration of the same concept: reconfigurable reaction networks act as templatefree recipes to specifically encode diverse glycan oligomers.
In the absence of a template, cells use a spectrum of mechanisms to limit glycan variability. At one end are quantitative kinetic mechanisms (Cardelli et al., 2018), such as the regulation of compartment residence times (Pantazopoulou and Glick, 2019), control of donorsubstrate levels (Parker and Newstead, 2019), or use of heteromeric enzymes to enhance sequential reactions (Varki et al., 2017, Chapter 4). At the other end are qualitative mechanisms that constrain which biosynthetic reactions are even allowed to occur: the selectivity of enzymes for their oligomer substrates, and the compartmentalization of enzymes within the Golgi. When enzymes are promiscuous, the kinetics of distinct reactions cannot be independently regulated, leading to variability (Biswas and Thattai, 2020). We have shown that, by splitting promiscuous enzymes across different Golgi compartments, this variability can be reduced or eliminated.
To synthesize oligomers without byproducts, cells must either decrease enzyme promiscuity, or increase the number of compartment types (Figure 1D). Both these strategies come at a cost, because there is a limit to the number of proteins a cell can encode. If all enzymes were highly selective, each with one specific substrate, a single compartment would suffice; but cells would require as many enzymes as oligomer types. On the other hand, an extensive system of membrane traffic proteins is required to maintain distinct Golgi compartments (Pantazopoulou and Glick, 2019). How do cells manage this tradeoff? Some GTase enzymes, such as those which synthesize the Nglycan oligomannose precursor in the ER, are highly selective and appear to act on just one or two oligomers; but most GTases are promiscuous, able to act on many oligomers or at many points on a single oligomer (Biswas and Thattai, 2020). This means having multiple compartments is crucial for limiting glycan variability in real cells. These ideas inform strategies for artificial glycan synthesis (Liu et al., 2019) and algorithmic selfassembly (Soloveichik and Winfree, 2007; Zeravcic and Brenner, 2014; Murugan et al., 2015): ‘stopandgo’ (Liu et al., 2019) or ‘stepassembly’ (Doty, 2012) approaches, which are analogous to multicompartment synthesis, expand the repertoire of synthesizable oligomers.
The regulation of glycan synthesis by compartmentalization has biological advantages. By redistributing enzymes within the Golgi, multicellular organisms can use the same set of enzymes to generate distinct glycan profiles in distinct cell types (Figure 2A; West et al., 2010; Fisher et al., 2019). The rapid and reversible changes in glycan profiles seen during infection and inflammation (Varki et al., 2017, Chapter 46) are more consistent with changes in enzyme localization than changes in enzyme expression. Such changes could also facilitate rapid evolution of glycan profiles in the context of hostpathogen interactions and speciation (Watanabe et al., 2020; Varki et al., 2017, Chapter 20). But this also means small errors in Golgi localization could be pathological: glycan perturbations promoting tumor invasiveness appear to arise due to errors in enzyme localization, not mutations in the enzymes themselves (Gill et al., 2013; and congenital glycosylation disorders are often correlated with defects in Golgi structure (Freeze and Ng, 2011).
The emergence of intracellular compartments was a watershed step in eukaryotic evolution (Dacks and Field, 2018). Many hypotheses have been advanced about the adaptive function of such compartments. Here, we have shown that the compartmental organization of the Golgi apparatus allows cells to control glycan synthesis despite enzymatic promiscuity, potentially explaining why this remarkable organelle is universally conserved across all extant eukaryotic lineages (Barlow et al., 2018). The ability to generate narrow, specific glycan profiles would have been advantageous to early eukaryotes (Wetzel et al., 2018), enabling the sophisticated intercellular interactions that underlie sex, cooperation and multicellularity.
Materials and methods
Methods are provided in the Appendices. Appendix 1: Definitions. Appendix 2: Proofs. Appendix 3: Detailed example of algorithmic growth.
Appendix 1
Definitions
Glycan oligomer
A set of monomers linked to form a finite tree. Oligomers grow one monomer at a time, so every oligomer or suboligomer (any subtree of the oligomer) has a welldefined root monomer and a welldefined direction of growth (Figure 1A).
Root chain
The path of specific linkages and monomers leading from the root to a given monomer (Figure 1A).
Acceptor monomer
A monomer in an oligomer that can be linked to a new donor monomer at some specific carbon, through the action of some GTase enzyme (Figure 1A).
Empty acceptor monomer
An acceptor monomer with nothing linked to any carbon, except the carbon on its root chain. A donor monomer becomes an empty acceptor monomer once it is linked to the oligomer (Figure 1A).
Branch
The full suboligomer on a given carbon of a given monomer. If nothing is linked to a given carbon, we say the corresponding branch is empty. New branches are initiated when empty carbons are linked to donor monomers (Figure 1A).
Tandem repeat
A chain in an oligomer that contains repeated instances of the same monomer type (Figure 1C).
Acceptorsubstrate
Rootsensitive enzyme: the acceptorsubstrate is a specific acceptor monomer type with an empty branch at the carbon to be linked, and a specific root chain. Branchsensitive enzyme: the acceptorsubstrate is a specific acceptor monomer type with an empty branch at the carbon to be linked, and specific branches or empty branches at all other carbons. Contextfree enzyme: the acceptorsubstrate is a specific acceptor monomer type with an empty branch at the carbon to be linked (Figure 1B).
Unidirectional acceptor block (branchsensitive enzymes)
The acceptorsubstrate of one enzyme is on a branch of the acceptorsubstrate of another, so that the action of the first blocks the action of the second (Figure 3B).
Bidirectional acceptor block (all classes of enzymes)
Two enzymes have the same acceptorsubstrate, so the action of the first blocks the action of the second, and viceversa. This is because the each enzyme can only act on the original unmodified acceptorsubstrate (for example, both enzymes act on the same carbon of the same monomer) (Figure 3B).
Compartment
A reaction compartment containing a set of specified GTase enzymes, and characterized by an average oligomer residence time (Figure 1A).
Input oligomer
An oligomer or suboligomer that is provided as an input to a compartment (Figure 1A).
Output oligomer
An oligomer that exits the compartment after some residence time, at some stage of growth (Figure 1A).
Series of compartments
An ordered set of compartment types in which every output of each compartment type is passed as an input to the next compartment type. An initial input is provided to the first compartment, and the last compartment produces the final outputs (Figure 1C).
Growth order
The order in which an oligomer is grown one monomer at a time, starting from a specific initial oligomer and leading to a specific final oligomer, through the action of successive enzymes in one or more compartments (Figure 3D).
Depthfirst growth order
A growth order in which, as soon as a new donor monomer is linked to an empty carbon of an acceptor monomer, the preexisting branches of that acceptor monomer no longer grow (Figure 3D).
Reaction network
The nodes of a reaction network represent distinct oligomers, and its directed edges represent singlemonomeraddition reactions. A reaction network shows all possible growth orders in a given compartment starting from a given input oligomer (Figure 2A).
Terminal oligomer
An oligomer with no outgoing edges in the given reaction network (Figure 2A).
Intermediate oligomer
An oligomer with at least one outgoing edge in the given reaction network (Figure 2A).
Trigger
An acceptorsubstrate that cannot be fully synthesized within a compartment starting from an empty acceptor monomer input (Figure 3D).
Runaway reaction
A reaction network, starting from a given input oligomer, that has at least one infinite path (Figure 2A).
Divergent reaction
A reaction network, starting from a given input oligomer, that has at least one fork beyond which reaction paths never reconverge (Figure 2A).
Algorithmic compartment
A compartment that has no linkage loops or acceptor blocks. At infinite residence times, for each possible input it specifically synthesizes a corresponding unique output. Algorithmic growth is the type of growth that occurs in an algorithmic compartment (Figure 3C).
Specifically synthesizable
A target oligomer is specifically synthesizable from an input oligomer if there is a series of one or more compartments that converts the input to the target as the unique final output, with no byproducts (Figure 1D).
Appendix 2
Proofs
Remark 1. Variability in assemblyline reactions
Consider a reaction compartment in which monomers are added, one at a time, to a growing oligomer. Assume monomers are in excess, and that oligomers grow independently of one another. The oligomer starts in some input state, proceeds through a series of transitions, and exits the reaction compartment after some residence time $T$. We model this as a continuoustime Markov process over a discrete, potentially infinite state space: each state is an oligomer configuration; each transition is an enzymecatalyzed singlemonomeraddition reaction, whose probability per unit time is proportional to the corresponding enzyme concentration. These states and transitions form an acyclic assemblyline reaction network. Given a reaction network starting from some input oligomer, define a truncated network by cutting off every reaction path at an arbitrary point (this should correspond to an oligomer height much larger than the input oligomer height plus the number of monomer types). The terminal oligomers of the truncated network are then either terminal oligomers of the original network, or truncated oligomers containing arbitrary numbers of tandem repeats. For any finite residence time $T$ the exit probability for any oligomer in the truncated network is nonzero. As $T\to \mathrm{\infty}$, or equivalently at high enzyme concentrations, the exit probability for any intermediate oligomer tends to zero, while the exit probability for any terminal oligomer or arbitrary tandemrepeat oligomer is nonzero. The input oligomer will be fully converted to a single specific final oligomer if and only if all the following conditions hold (Table 1): (a) The compartment has no runaway reactions so there are no arbitrary tandem repeat oligomers; (b) The compartment has no divergent reactions so every input leads to a unique terminal oligomer; (c) The compartment has a sufficiently long residence time (or equivalently, sufficiently high enzyme concentrations) so only this unique terminal oligomer exits as an output.
Remark 2. Constructing a compartment’s linkage network
Linkage networks are only defined for contextfree and branchsensitive enzymes; for these enzyme classes the fate of a bare monomer does not depend on its position in a tree. The nodes of a linkage network represent distinct monomer types or triggers; its directed edges represent acceptortodonor linkages at specific carbons. (a) To construct the linkage network of a compartment with contextfree enzymes, we add an arrow from one monomer type to another if the corresponding acceptortodonor linkage can be carried out by some enzyme in the compartment. (b) To construct the linkage network of a compartment with ideal branchsensitive enzymes is more involved. We first add an arrow from one monomer type to another if the corresponding acceptortodonor linkage occurs on any oligomer that can be synthesized starting from any empty acceptor monomer input. We need consider only oligomers whose height is less than or equal to that of the tallest acceptorsubstrate of any GTase enzyme in the compartment. An enzyme whose acceptorsubstrate is a trigger will not be represented among the arrows we have added so far. We must explicitly list each trigger and draw an arrow from its acceptor monomer to the relevant donor monomer type. A trigger is effectively a novel monomer type in the linkage network, such that other donor monomers can be added to it, but it cannot be added to other acceptor monomers. This gives the full linkage network of the compartment.
Lemma 1
Runaway reactions ⇔ linkage loop.
Proof
Consider a reaction network starting from some input oligomer. Keep all enzymes involved in this reaction network, remove other enzymes from the compartment. Suppose the reaction network has an infinite runaway path. Each reaction corresponds to the addition of one monomer to an oligomer. Therefore the reaction network contains at least one oligomer with an arbitrarily long roottotip chain. Since the number of monomer types is finite, the chain must include at least two instances of the same monomer type added within the compartment. Therefore the compartment’s linkage network contains a loop. Conversely suppose a compartment’s linkage network contains a loop. Then there is at least one monomer type, added at some step of the reaction network, on which a branch can be grown that includes another instance of the same monomer type. This process can be iterated ad infinitum to produce arbitrary tandem repeats. Therefore, the reaction network contains an infinite runaway path. (This argument is reminiscent of the pumping lemma for formal languages.) Runaway reactions are not possible with ideal rootsensitive enzymes, since they act at a defined depth from the root monomer (Figure 3A).
Lemma 2
Divergent reactions ⇒ acceptor block.
Proof
Consider a reaction network starting from some input oligomer. Keep all enzymes involved in this reaction network, remove other enzymes from the compartment. Suppose the network has a divergent reaction. A fork in a reaction network occurs when two enzymes can act on the same oligomer. If the enzymes could act in either order the fork could immediately reconverge. Therefore, there is at least one pair of enzymes such that the action of the first enzyme blocks the subsequent action of the second. (a) For rootsensitive and contextfree enzymes, there is only one way a divergent reaction can occur: both enzymes compete to act on the same carbon of the same monomer (bidirectional acceptor block). In this case, acceptor blocks are necessary and sufficient for a divergent reaction: there is no way for the reaction paths to reconverge. (b) For branchsensitive enzymes, there are two ways a divergent reaction can occur: both enzymes have the same acceptorsubstrate (bidirectional acceptor block) or the acceptorsubstrate of the first enzyme is on a branch of the acceptorsubstrate of the second (unidirectional acceptor block). In this case, acceptor blocks are not sufficient for a divergent reaction, as the reaction paths might reconverge via the action of a subsequent enzyme (Figure 3B).
Lemma 3
If a compartment has a reaction network, starting from some input oligomer, that is finite (no linkage loops) with multiple terminal oligomers (due to acceptor blocks). Then there is an acceptorblockfree subset of the original enzymes that specifically synthesizes one of the original terminal oligomers from the input oligomer.
Proof
Consider compartment with a finite reaction network starting from some input oligomer, with multiple terminal oligomers. Keep all enzymes involved in this reaction network, remove other enzymes from the compartment. Assume infinite residence time, so every possible growth order starting from the input oligomer reaches some terminal oligomer. Now there is at least one growth order in which a given enzyme involved in an acceptor block is completely blocked from acting because its acceptorsubstrate or a branch of its acceptorsubstrate is modified by some other enzyme. This growth order is retained when we remove the blocked enzyme. By iterating this procedure we eliminate every acceptor block. The resulting compartment is algorithmic (no linkage loops or acceptor blocks), so every growth order leads to just one of the original terminal oligomers (by Lemma 1 and Lemma 2). Removing blocked enzymes in a different order may select a different terminal oligomer; and not all terminal oligomers can necessarily be selected in this way.
Lemma 4
All growth orders in an algorithmic compartment satisfy the following properties, which we collectively characterize as ‘algorithmic growth’. (a) Contextfree enzymes: Identical empty branches of identical monomers are identically extended. (b) Ideal branchsensitive enzymes: Identical empty branches of identically branched monomers are identically extended; and growth is depthfirst. (c) Ideal rootsensitive enzymes: Every growth order to a given terminal oligomer from any suboligomer is algorithmic.
Proof
By definition, algorithmic compartments have no linkage loops or acceptor blocks. Therefore the reaction network starting from any input is finite, with a single terminal oligomer. Since there are no bidirectional acceptor blocks and the final oligomer is terminal, identical acceptorsubstrates (defined separately for each class of enzymes) are identically extended at the same branch, and have identical fates in the final oligomer. (a) Contextfree case: acceptorsubstrates are some monomer type. (b) Branchsensitive case: acceptorsubstrates are some monomer type having or lacking specific branches. In addition, since there are no unidirectional acceptor blocks, once any enzyme acts on its acceptorsubstrate its preexisting branches no longer grow. This implies growth is depthfirst. (c) Rootsensitive case: acceptorsubstrates are some monomer type with a specific root chain. Since every monomer in an oligomer has a distinct root chain, every growth order to a given terminal oligomer from any suboligomer is algorithmic.
Lemma 5
A target oligomer can be specifically synthesized from an input oligomer in a single compartment if and only if there is an algorithmic growth order from the input to the target.
Proof
Suppose a target oligomer can be specifically synthesized from an input oligomer in a single compartment. The reaction network starting from the input oligomer must have the target as its unique terminal oligomer, so there are no linkage loops. We can retain an acceptorblockfree subset of enzymes that specifically synthesizes this terminal oligomer from the input (Lemma 3). The resulting compartment is algorithmic, therefore any remaining growth order is algorithmic (Lemma 4). Conversely, suppose there is an algorithmic growth order starting from the input, with the target as the final oligomer. Each step of the growth order corresponds to the action of some GTase enzyme. Suppose we construct a compartment with just these enzymes. This will certainly synthesize the final oligomer from the input. However, we must check whether this set of enzymes generates runaway and divergent reactions that lead to the synthesis of tandem repeats or terminal oligomers other than the desired target. By assumption, identical empty branches on identical acceptorsubstrates are identically extended, and therefore have identical fates in the final oligomer. This implies there are no bidirectional acceptor blocks. This also means no empty branch of the final oligomer can be extended by any enzyme, so it is terminal and there are no linkage loops. In the branchsensitive case growth is depthfirst, meaning no branch of the acceptorsubstrate of one enzyme is later extended by another enzyme, so there are no unidirectional acceptor blocks. Therefore, the constructed compartment is algorithmic. At infinite residence times, it will specifically synthesize the target oligomer from the input oligomer.
Theorem 1
A target oligomer can be specifically synthesized from an input oligomer if and only if it can be specifically synthesized from that input oligomer in a series of singleenzyme compartments.
Theorem 2
A target oligomer can be specifically synthesized from an input oligomer in a series of $N$ compartments if and only if there is a growth order from the input to the target that can be fully decomposed into $N$ algorithmic growth stretches.
Proof
We prove both theorems together. Suppose the target oligomer can be specifically synthesized from an input oligomer in a series of $N$ compartments (Stage I, Figure 4A). The outputs of each compartment are passed as inputs to the next compartment. Every possible growth order starting from the initial input oligomer leads to the target as the unique final oligomer, so we know there are no arbitrary tandem repeats generated in any compartment, and therefore no linkage loops. A subset of these growth orders will remain if each compartment has infinite residence time (Stage II). Each reaction network starting from each input of each compartment is finite. Therefore, we can replace the enzymes in each compartment with an acceptorblockfree subset that specifically synthesizes one of the original terminal outputs from any of the original inputs (Lemma 3). This leaves a set of $N$ algorithmic compartments (Stage III). All remaining growth orders start from the initial oligomer, pass through just one terminal oligomer of each successive compartment, and produces the target oligomer as the unique terminal output of the last compartment. Theorem 1: Among the growth orders at Stage III, since enzymes act in every possible permutation, there is at least one growth order in which each given enzyme acts successively on every available instance of its acceptorsubstrate on the oligomer, before the next enzyme acts. This growth order is retained even once we replace each compartment by a series of singleenzyme infiniteresidencetime compartments (Stage IV). The converse is trivial. Theorem 2: Since every compartment is algorithmic at Stage III, any growth order within each compartment is algorithmic (Lemma 4). Conversely, suppose there is a growth order from the input to the target that can be fully decomposed into $N$ algorithmic stretches. Then each stretch can be achieved within a single compartment (Lemma 5) so the target oligomer can be specifically synthesized from an input oligomer in a series of $N$ compartments.
Appendix 3
Detailed example of algorithmic growth
We want to test (Appendix 3—figure 1A) if a desired target oligomer (full structure, filled and empty circles) can be specifically synthesized from a given input oligomer (filled circles only) in one compartment. This is possible if and only if there exists an algorithmic growth order from the input to the target (Appendix 2, Theorem 2). We can already see that identical empty branches of different instances of monomer type $A$ in the input oligomer (red) have distinct fates in the target oligomer, so algorithmic growth is not possible using contextfree enzymes. So we look instead for a branchsensitive solution.
We show examples of possible growth orders for two suboligomers (Appendix 3—figure 1A, top). At each growth step an empty circle becomes filled as a new monomer is added. Depthfirst growth means no new branch can be initiated on a monomer with incomplete existing branches; the leftmost vertical arrow is not a depthfirst step. Identical acceptorsubstrates must be identically extended at the same branch; therefore if two growth paths converge to the same acceptorsubstrate, they must have the same subsequent growth step.
The input oligomer contains many distinct acceptorsubstrates, each potentially in multiple copies, and each with a desired terminal fate (labeled with a Λ). A few examples of these are shown here (Appendix 3—figure 1B). The terminal fates of empty monomers ($\widehat{A}$, $\widehat{B}$, $\widehat{C}$, $\widehat{D}$) are of particular interest, since these are the allowed fates of any newlyinitiated branches. Under depthfirst growth it is sufficient to consider how new branches are initiated on acceptorsubstrates whose existing branches are already terminally extended. Instead of a graphical representation (Appendix 3—figure 1B), we use a recursive representation showing the branches linked to each carbon of each acceptormonomer type (Appendix 3—figure 1C). Thus, oligomer $\widehat{A}$ is monomer $A$ linked to branches $\widehat{B}$, $\widehat{C}$, $\widehat{D}$ on its three carbons. We list the set of distinct initial acceptorsubstrates (bold boxes colored by acceptor monomer type) and their desired terminal fates in the target oligomer (Λ). Boxes are colored according to acceptor monomer type; each slot shows any existing terminallyextended branches; empty carbons are labeled '−'. We have not shown the trivial case of monomer type $D$.
For branchsensitive enzymes, an algorithmic growth order is essentially determined by the choice of branch order. (Depthfirst growth is enforced by assumption, since we consider only acceptorsubstrates with terminallyextended existing branches.) We must find a single consistent branch order for each monomer type, such that each distinct acceptorsubstrate achieves its desired terminal fate; we need consider only branches that are actually observed in the target oligomer. The initiation of all possible branches in all possible orders is represented as a transition graph (Appendix 3—figure 1D). Each node of the graph represents distinct acceptorsubstrates, using the box notation from Appendix 3—figure 1C. Each directed edge represents the initiation and terminal extension of a branch on an empty carbon. Bold colored arrows show a possible choice of successive branch additions, from initial acceptorsubstrates (bold boxes) to desired terminal fates (Λ). There can be no bold outward arrows from terminal fates; the bold selfloop represents an acceptorsubstrate that is already at its desired terminal fate. By the definition of algorithmic growth, there can be only one bold outward arrow from each acceptorsubstrate. Any acceptorsubstrate not reachable from an empty monomer is a trigger. The branch order search might have a single unique solution, as with monomer type $C$; or multiple solutions, as with monomer type $B$ (only one solution is shown here). There may be no solutions, since each choice of branch initiation cuts off other paths. In this example, there is no set of bold arrows that simultaneously achieves all desired terminal fates for monomer type $A$. For the choice of arrows shown here, all desired terminal fates are achieved except for that of the empty monomer $A$. Therefore, the given target cannot be specifically synthesized from the given input in a single compartment using branchsensitive enzymes. We leave it as an exercise for the reader to show that the target oligomer can be specifically synthesized from the input oligomer by branchsensitive enzymes in two compartments.
Data availability
Matlab source code has been provided for generating plots in Figure 2B.
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Decision letter

Patricia BassereauReviewing Editor; Institut Curie, France

Naama BarkaiSenior Editor; Weizmann Institute of Science, Israel

Alberto LuiniReviewer; National Research Council, Italy
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
Acceptance summary:
The manuscript addresses the interesting question of how, despite the sloppiness and promiscuity of the glycoenzymes, a limited variety of glycans are produced in the Golgi. The authors develop a complete formal theoretical framework and show that the subGolgi compartmentalization is key. It allows distributing the enzymatic activities through multiple processive Golgi compartments which effectively reduces the variability in the glycan output.
Decision letter after peer review:
Thank you for submitting your article "Golgi compartments enable controlled biomolecular assembly using sloppy enzymes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Naama Barkai as the Senior Editor.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Summary:
The manuscript addresses the question of the control of glycan variability in the Golgi achieved by sloppy glycoenzymes from a theoretical perspective. The authors propose that the variability issue can be solved by a spatial segregation of the enzymes, which could a posteriori provide an explanation for the functional origin of the Golgi compartmentalization.
Essential revisions:
The three reviewers and myself think that your work could be of strong interest for biologists working on membrane traffic and specially on Golgi, but in its current form, the message and the predictions for practical tests on living cells are not easily accessible to the majority of eLife readers. Therefore, we consider that you should deeply revise your manuscript to address this serious concern and discuss a few practical cases. In addition, since the dynamical structure of the Golgi is still a matter of debate, we think that you should add a discussion on how your model depends on Golgi maturation, on the consequence of a continuous gradient vs discrete distribution of the enzymes, on the impact in a multiplecompartments vs. a single compartment system. Eventually, we would like that you discuss the robustness of the model to the "uniform depthfirst growth" rule.
[Editors' note: further revisions were suggested prior to acceptance, as described below.]
Thank you for submitting your article "Golgi compartments enable controlled biomolecular assembly using promiscuous enzymes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Naama Barkai as the Senior Editor. The reviewers have opted to remain anonymous.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Summary:
This manuscript addresses theoretically the question of the control of glycan variability in the Golgi achieved by sloppy glycoenzymes. The authors propose that the variability issue can be solved by a spatial segregation of the enzymes, which could a posteriori provide some functional origin to the Golgi compartmentalization.
Essential revisions:
The reviewers have appreciated your effort to facilitate the accessibility of your work to biologists reading eLife articles in your revised version. Nevertheless, we think that your paper could have a much stronger impact among biologists if you could further modify the presentation. We recommend following suggestions from reviewer #2 (see below).
Reviewer #2:
What I like about the work is the element of bringing in a new dimension into the intricate biological processes in the Golgi that involve glycan synthesis, binding, and selfassembly. However, I find the choice of language in the manuscript seriously concerning. The authors have chosen to present their main idea as mathematical theorems supported by proofs and lemmata. If the aim for this choice were to give the readers the assurance that what they are seeing is unquestionable fact and not just the opinion or suggestion of theoreticians, I believe this could be done in a much more accessible way. If the aim were to purport that the authors are making a new contribution to the field of mathematical logic by proving previously unknown theorems, then the aim would be incorrect and inappropriate. This has already been done in the specific context of algorithmic selfassembly in the papers that the authors cite themselves. The authors are merely applying these ideas to the specific biological problem at hand.
The main tenet of the authors' argument is enumerating the number of mutually exclusive and complementary possibilities in polymerization. This is basically equivalent to saying that a coin has two sides. One can, of course, make this into a theorem in mathematical logic and provide a proof of it, which can take pages and pages. However, what a normal user of this theorem would want to know from it will only be that we have only two mutually exclusive possibilities when we are throwing a coin: heads and tails. If we throw a dice, we have six possibilities. That's it. I do not see why the authors have chosen the rather heavy mathematical language to motivate the use of this simple enumeration of possibilities, for this particular biological application.
In response to my previous recommendation to streamline the presentation and make it more accessible to the appropriate biological readership of the journal, the authors moved some of the theorems and proofs to the appendix. As a result, the main manuscript now starts from lemma 3 and then wanders into some other mathematical blocks along the way. The only biological analysis is done in the captions of the figures, which are excessively long. Figure 1's caption is 29 lines if I managed to count correctly.
I think as it stands the manuscript will likely not attract its full potential in readership because of this confused style. My suggestion to the authors is to remove the mathematical language altogether and write the manuscript as if they are applying the enumeration argument to the biological process. They can motivate it, provide the examples next, and conclude by arguing for the generality of the principles and the arguments. I imagine this would make a wonderful paper that is also going to be read by the readers of eLife.
I cannot recommend publication of the manuscript in its current style, not even with the revision.
https://doi.org/10.7554/eLife.49573.sa1Author response
Essential revisions:
The three reviewers and myself think that your work could be of strong interest for biologists working on membrane traffic and specially on Golgi, but in its current form, the message and the predictions for practical tests on living cells are not easily accessible to the majority of eLife readers. Therefore, we consider that you should deeply revise your manuscript to address this serious concern and discuss a few practical cases.
We have taken very seriously the input that our work should be more accessible to biologists. Before preparing our revision, we circulated the manuscript to cell biology colleagues to solicit specific feedback on (a) relevance of results and (b) clarity of exposition. Based on this feedback and on the reviewer comments, we have made major revisions to the paper, summarized here:
New emphasis: We received feedback that our analysis of promiscuity is potentially the most useful direct result of our work. We have therefore revised the manuscript to focus deeply on the causes and consequences of enzyme promiscuity. Both in the title and throughout the text, we have replaced the word “sloppy” (which was borrowed from the selfassembly literature) with the word “promiscuity” which is more informative and relevant to biologists. We have added several paragraphs to the Discussion, in which we outline the tradeoff between promiscuity and compartmentalization.
New results: We have added completely new results, which compare the behavior of two classes of enzymes that span a range of possible promiscuity: the maximally promiscuous “branchinsensitive enzymes” and the less promiscuous “branchsensitive enzymes”. This includes two new Lemmas (3 and 4) in Supporting Information. Our discussion is updated to show how promiscuity and compartmentalization jointly influence glycan variability, with several new predictions and new examples.
Modified figures: We have modified all figures (Figures 1, 2, 3 and Appendix 3—figure 1) to update our comparison of branchinsensitive and branchsensitive enzymes. Our key new results are summarized in an example discussed in Figure 3C.
Streamlined text and terminology: We have replaced several technical terms with more informative terms and added multiple paragraphs that provide a nontechnical background to the mathematical theorems. We have replaced the term “uniform depthfirst growth” with “algorithmic growth” and expanded its definition to apply to both branchinsensitive and branchsensitive enzymes (Lemma 4).
In addition, since the dynamical structure of the Golgi is still a matter of debate, we think that you should add a discussion on how your model depends on Golgi maturation, on the consequence of a continuous gradient vs discrete distribution of the enzymes, on the impact in a multiplecompartments vs. a single compartment system.
We thank the reviewers for pointing out this lacuna in our discussion. We have added new text (in the section “Glycan synthesis by promiscuous, stochastic enzymes”) that discusses in detail the implications of the transport and maturation models of the Golgi. We discuss why, as long as the maturation transitions are sufficiently rapid, our results will apply to both models. We have noted that our results will not apply to the case of a gradient of enzymatic compositions.
Eventually, we would like that you discuss the robustness of the model to the "uniform depthfirst growth" rule.
We thank the reviewers for this insightful comment. Indeed, the definition of “uniform depthfirst growth” is not universal but depends on the nature of the underlying enzyme promiscuity. In our revision we have explicitly derived independent results for two classes of enzymes that span a range of promiscuity: branchinsensitive and branchsensitive enzymes. We have replaced the term “uniform depthfirst growth” (which applies only to the latter class) with the broader term “algorithmic growth” and described the different types of algorithmic growth which apply to the different enzyme classes (Lemma 4). Our main result (Theorem 2) still applies, to both classes of enzyme promiscuity. Since real GTase enzymes will have intermediate promiscuity between the two classes, our results therefore provide both an upper and a lower bound on the number of compartments needed to specifically synthesize a given oligomer (Figure 3C). We therefore expect our predictions to be applicable in many cellular contexts.
[Editors' note: further revisions were suggested prior to acceptance, as described below.]
Essential revisions:
[…]
Reviewer #2:
What I like about the work is the element of bringing in a new dimension into the intricate biological processes in the Golgi that involve glycan synthesis, binding, and selfassembly. However, I find the choice of language in the manuscript seriously concerning.
We thank the reviewer for this suggestion. We have extensively revised the figures and text of the manuscript. We have streamlined the flow of text to focus on just the key ideas and remove distracting technical asides.
The authors have chosen to present their main idea as mathematical theorems supported by proofs and lemmata. If the aim for this choice were to give the readers the assurance that what they are seeing is unquestionable fact and not just the opinion or suggestion of theoreticians, I believe this could be done in a much more accessible way.
This is an important point. We had chosen to present our results in the form of theorems, rather than the alternative possibility of simulations. Our aim was threefold. First, we hoped to highlight general lessons, which is extremely difficult using simulations since any implementation of a simulation involves making several specific choices. Second, we wanted to ensure that all our assumptions were clearly stated, and that we did not miss any hidden assumptions about the behavior of enzymes. Third, we sought to provide a bridge to selfassembly researchers, who we hoped would be stimulated by ideas derived from this compelling biological example of controlled synthesis. We hope our updated text, involving more examples to buttress the mathematics, is less distracting and provides a more accessible exposition of the central ideas.
If the aim were to purport that the authors are making a new contribution to the field of mathematical logic by proving previously unknown theorems, then the aim would be incorrect and inappropriate. This has already been done in the specific context of algorithmic selfassembly in the papers that the authors cite themselves. The authors are merely applying these ideas to the specific biological problem at hand.
Although the selfassembly literature has a rich body of mathematical theorems, to our knowledge there is no specific application of these ideas to the construction of treelike objects, which glycans are an example of. The basic idea of variability (runaway and divergent reactions) has been previously explored. However, the theorems we have proved go beyond application of existing theorems, and to our knowledge they are not to be found elsewhere the literature.
The main tenet of the authors' argument is enumerating the number of mutually exclusive and complementary possibilities in polymerization. This is basically equivalent to saying that a coin has two sides. One can, of course, make this into a theorem in mathematical logic and provide a proof of it, which can take pages and pages. However, what a normal user of this theorem would want to know from it will only be that we have only two mutually exclusive possibilities when we are throwing a coin: heads and tails. If we throw a dice, we have six possibilities. That's it. I do not see why the authors have chosen the rather heavy mathematical language to motivate the use of this simple enumeration of possibilities, for this particular biological application.
One of our major results is, indeed, the enumeration of mutually exclusive possibilities. The reviewer is correct that the application of this result does not require the detailed machinery of the theorem. However, one of our key contributions (summarized Table 1) is to show that the possibilities we have listed are indeed comprehensive. To prove we have not missed out other sources of variability, we required the full logical machinery. However, we agree that these details need not be in the main text. We have restricted them to the Appendices.
In response to my previous recommendation to streamline the presentation and make it more accessible to the appropriate biological readership of the journal, the authors moved some of the theorems and proofs to the appendix. As a result, the main manuscript now starts from lemma 3 and then wanders into some other mathematical blocks along the way. The only biological analysis is done in the captions of the figures, which are excessively long. Figure 1's caption is 29 lines if I managed to count correctly.
We have made major changes to the flow of the text, with the inclusion of new examples, new figure panels, and reordering of key results. We have removed the distracting technical excursions, instead we now motivate the result with several examples, before moving on to their mathematical basis. We agree the figure captions were too long in the previous version. We have reorganized the text, so the captions contain only details needed to interpret the figures, while the text contains fuller discussion and examples.
I think as it stands the manuscript will likely not attract its full potential in readership because of this confused style. My suggestion to the authors is to remove the mathematical language altogether and write the manuscript as if they are applying the enumeration argument to the biological process. They can motivate it, provide the examples next, and conclude by arguing for the generality of the principles and the arguments. I imagine this would make a wonderful paper that is also going to be read by the readers of eLife.
We thank the reviewer for taking this broad view of the manuscript. We have now done exactly this, started with examples, provided some intuition for them, and concluded with arguments for generality. All the material needed to see what’s “under the hood” is still contained in the Appendices for the technical reader, and we have directed readers to the relevant Lemma or Theorem at appropriate points in the text.
https://doi.org/10.7554/eLife.49573.sa2Article and author information
Author details
Funding
Simons Foundation (287975)
 Mukund Thattai
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We thank Ajit Varki for introducing us to glycans, and Arnab Bhattacharyya for helping us view glycosylation through the algorithmic lens. We thank Ansuman Biswas, Ramya Purkanti, Somya Mani, Mugdha Sathe, Sachit Daniel, Kabir Husain and Amit Singh for useful discussions. We thank Kaadambari for critical inputs during writing.
Senior Editor
 Naama Barkai, Weizmann Institute of Science, Israel
Reviewing Editor
 Patricia Bassereau, Institut Curie, France
Reviewer
 Alberto Luini, National Research Council, Italy
Version history
 Received: June 21, 2019
 Accepted: June 28, 2020
 Accepted Manuscript published: June 29, 2020 (version 1)
 Version of Record published: July 14, 2020 (version 2)
Copyright
© 2020, Jaiman and Thattai
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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