TIG - - March 1987. VoW. 3, no. 3

A question that has fascinated molecular giycobiologists for the last two decades is why the carbohydrate moieties of glycoconjugates dmnge in structure dm:ing development, differentiation and transforma- tion o f m a l n m a l i a n t i s s u e s 1. These changes affect carbo- hydrates associated with glyco- protei~-~ gly~olipids and pro- teoglycans of widely different biochemical function and yet they are completely compatible with cell viability. It seems likely that the expression of a new array of carbohydrate structures at the cell surface is required for the acquisition of certain cellular properties. However, this functional role for carbo- hydrates must occur independently and within the framework of the basic structural role that carbo- hydrates play to maintain the conformation, localization and biological activity of the individual glycoconjugates to which they are attached. Two general functions for carbohydrates must therefore be envisaged: an intramolecular function that is specific and potentially variable for each glycoconjugate, and an @.*.-rmo/ecu/ar function reflected in carbohydrate changes that often affect more than one class of glycoconjugate

The two interdependent roles of carbohydrates, coupled with the fact that they exist mainly in covalent association with protein or lipid, mean that d e f i n i n g functional relationships for carbohydrates is difficult. In addition, though carbohydrate moieties are synthesized from about ten different sugars, their structures are complicated because of the variety of linkages that may occur between sugar residues. Since it is clear that a change in a single sugar residue will affect not only the composition but the overall conformation of a carbohydrate, the hmctional roles for carbohydrates in recognition phenomena are potentially enormous. Fortunately, the last ten years have seen revolutionary advances in our abilities to determine carbohydrate structures precisely ~'a and in the development of methods for describing the conformation of complex carbohydrate moieties in solution 4. Several laboratories have also reported the isolation, from a variety of cell lines, of mutants that exhibit specific alterations in carbohydrate biosynthe- sis s'~ and so a classic genetic approach to investigating carbohydrate function has become feasible. Giyco- sylation mutants that express new glycoconjugates have proved invaluable not only for functional studies but also for defining the pathways of carbohydrate biosynthesis in mammals. In this article I wi~ sununarize the selection and biochemical characteriza- tion of animal cell giycosylation mutants and outline the usefulness of the mutants in defining functional roles for carbohydrates.

Select ion and proper t ies ofglycosylat ion mutants

Lectins, which are carbohydrate-binding proteins from plants, have proved to be excellent selective agents for obtaining giycosylation mutants 7. They bind to specific sugars with a range of affinities that depend

1987, Else~.r Pul~aUou~ C~n~ddee 01¢~ - 9525/ST~J02 eO

review G lycosylation mutants and

the functions of mammalian carbohydrates

Pamela Stanley Glycosylation mutants o/mammalian ceils synthesize novel carbohydrate structures that are expressed on ¢ellulag glycoconjugates. Such mutants provide many avenues for inues~afng the fu~ctio~ of carbohydrates, both m @eir effects on the behavior of individual macromole.cul~ (endo~nous or introd~ed into the cell) and on the array

of structures expressed at ~bo surface of ceils.

on the configuration of the sugar, and they therefore interact with a broad spectrum of the carbohydrate moieties at the cell surface. In addition, several lectins are cytotoxic ior cultured cells, allowing direct selection for lecfin resist~ce. Alternative selections, such as radioactive-sugar-suicide or screens for altered sugar incorporation, often give rise to the same mutant types that are obtained with lectins s. This is also true for selections aimed at specific glycoprotein membrane receptors, although giyco- sylation mutations that Zter the expression of the mannose 6-phosphate receptor or the low density lipoprotein (LDL) receptor appear to be a fairly uncommon subset of the giycosylation mutants obtained with direct lectin selections from Chinese hamster ovary (CHO) cells s'9.

Lectin resistance can occur for a variety of reasons and even those related to g]ycosy[ation may be many and varied. For example, eight of the giycosylation mutants obtained in CHO cells are resistant to wheat germ agglutinln and yet each has a different giycosylation defect e. Fortunately, all glycosyiation mutants isolated to date show characteristic cross- resistances or hypersensitivities to lectins of different carbohydrate-binding specificities. Thus isolates can be phenotypicaliy categorized by comparing their resistance to or binding properties for a pane[ of lectins ~, then the mutants can he distinguished genotypically by, ,mplementation using somatic cell hybrids 7. This m~alysis is critical for determining whether lelated or apparently identical phenotypes arose from mutation(s) in the same or different genes. Biochemical analysis is greatly aided by knowing whether a mul~mt represents a new genotype.

Detailed biochemical studies of a putative glyco- sylation mutant usually begin with structural analysis of the carbohydrate to determine the new carbohy- drates synthesized by the mutan0 °. Radiolaheled ~ycepeptid~.',s may be fractiouated according to size, ¢~rge or t'~eir differential af~nlties for lectins. The species tt, be structurally characterized are identified by differences between giycopeptide profiles of the mutant and parent cells. This may be achieved either indirecdy, by c.omp~ring with standards after sequc.'~.,ally removing each residue with a glycosidase or 4i~r~Cr~b. by a physical method such as XH-NMR spec~o~:opy. Once a particular sugar residue or linkage is implicated as the basis of a mutant phenotype, assays to measure the reactions involved in generating that structure are established. A difference in a specific enzyme or transport activity

r iews that is eyaL, ibited by independent mutant isolates of identical phenotype is considered to provide the biochemical basis of the mutation.

The biochemical reactions in N-linked carbohydrate biosynthesis that are altered in each of 22 animal cell mutant types are shown in Figs 1 and 2. Recessive mutants, typified by the loss of an activity and the accumulation of a truncated or inu~ature structure, are defective in the reactions that are circled. Dominant mutants, typified by the acquisition of an

0 ~ '~ ' • D e°=-:"ntP-- • O 6DPM I --- . . . . : o - I - I I - D

GIc;NAc-T G I c M A c - T BII,4IMon - T

f 6OPM.IMon- T's

l

°'- o_,,_,._iD 0 0 0

D.P @ I " (I) 5DPM~=~ImdDoI-P-M I Uon-T'|

DoI-P-M v Synlhelnse ~ ( ~ )

o-- o,,. o o-o-" " o - a - m - o 0--0 --0 / D,~ 1 " ® ,

GDPr.Lcp~eD01-P-~Ic L SIc-T.; Ool-P-GIc • SynthlllClSe o-.o~.

°- -°~ °'~" o - U - I - D A - A - A - o - o - o " .~ ®

ProteJnlOhgosocchoryO-T

° - ° - ° " o-u-II-Asn [PROCESSINGI A - A - A - o - o- o ~ / I

Glucosodn|eZ

• A.,,~Glu©osldosell) 'n' ~) O-- o.~ • -~ o-O ~ ~ o -m-m-Ash

° - ° "°~o'~,onno,.des, j[,) Z (~)

~ - - ° ~ o - i i - n - A o n

O U~PGn +.~icNAc-T Ir ~)

o ) o - u - n - A S h - Hybr,d

B - O 0 o , ~ u o n n o s n d o | e l s ) ~ )

II : ~ o - I I - l - A s n

-UDPGn +GIcNAc- Tn'

~8) I I - ° " o - i - I I - A s n GDPM t& -- ~GOPFur.... ,#" l - ° s [ ' ~ GleN&c- TIZ

• . 4ba~, . . . . . / UOPGn~L..GIcNA¢ -T u0pG.-~ m B~,, Z ........... ,;~-e(I~)Fvc- T 1 ~ )

. - o . ~ o m - o . : i ~ - ~ o - O - . - A s n i1_ o . . o - - I I - / - l l n i i _e . , o - I I - U - A l n

Biealeanery Breached Biseeled

Fig. 1. Synthesis of N-iinked carbohydrates. The Teactions and en; , .~s involved in N-linked carbohydrate biosynthesis in mammalian cells are summarized usang the following symbols and abbreviations: D = dotichylpymphosphate; Dol = dolichol; T = tramferase; Glc, & = Glucose; Gn, GIcNAc, [] = N-acetylglucosamins; M, Man, O = Mannase; Gal, A = galactose and Fuc, 4) = fucose. The reactions known to be b/oched m animal cell glycosylation mutants are indicated by a slash ~nd a circled number, while the transferase activity that has been shown to increase is indicated by a square. All of these mutants as well as some others that might produce altered

?~carbohydratss have been reviewed in detail elsewhere 5'6,10.

TIG - - M a r c h 1987, VoL 3, no. 3

activity and the synthesis of novel carbohydrates, are affected in reactions designated by a square. Many of these lesions have been described independently by several laboratories in mutants isolated from different cell lines s'6. Several of the mutations that affect N- linked biosynthesis would also be expected to modify O-linked carbohydrates as well as the carbohydrates associated with glycolipids and proteoglycans. In contrast, recently described proteoglycan mutants appear to be affected in reactions specific for the synthesis of proteoglycans la.

Although in most cases a change in a specific glycosyltransferase or other enzyme activity has been correlated with a mutant phenotype, in none of these cases is it known whether the mutation affects a structural or a regulatory gene. It is therefore important to keep selecting new mutants with the aim of obtaining more mutant alleles. For example, LeclA CHO mutants possess a N-acetylglucosamine trans- ferase I (GIcNAc-TI) activity that has hif)ter apparent Km values for both UDP-GIcNAc and carbohydrate substrate, strongly suggesting that the LeclA mutation resides in the structural gene for GlcNAc- TI ]4. The Lecl CHO mutation, thou.~h it abolishes GIcNAc-TI activity, belongs to the san~e complemen- tation group as LeclA, and therefore would also be expected to affect the structural gene for GIcNAc-TI. Molecular analysis of the GIcNAc-TI genes in Lecl and LeclA cells should reveal different nucleotide changes which could be correlated with the observed effects on enzyme activity. Sinfilarly, Lecl3 and Lecl3A CHO mutants represent alternative alleles at the GDP-mannose-4,6-dehydratase locus ts.

With the exception of the lesion labeled 11, the mut.:fions s~mmarized in Fig. 1 would be expected to alter only N-linked carbohydrates. However, the mutations affecting the terminal glycosylation re- actions summarized in Fig. 2 would be expected, in many cases, to change the structure of O-linked moieties on glycoproteins as well as the carbohydrates of glycolipids and proteoglycans. For example, mutations that reduce the level of a nucleotide sugar in a particular cellular compartment (e.g. mutations 14, 15 and 18 in Fig. 2) and mutations that reduce or increase the activity of a glycosyltransferase (e.g. mutations 16, 17, 19, 20, 21 and 22 in Fig. 2) shonld affect all glycoconjugates that usually contain that sugar. To add to me potential complications in the structural changes stemming from a glycosylation mutation, ff there are limiting amounts of a substrate (such as a nucleotide-sugar) or if an enzyme or transport activity is limiting, this may differentially affect particular classes of glycoconjugates depending on K,, differences between substrates. Detailed analyses of the carbohydrates synthesized by glycosylated mutants and their revertants should help to define the factors that affect the direction of glycosylation pathways.

Uses ofglycosylat ion mutants A mutant cell line affected in a single glycosylation

reaction is invaluable for biochemical, genetic and functional studies of mammalian carbohydrates (Table 1). In the rest of the article, ! will outline the g, meral areas m which glycosylation mutants have already contributed new information and suggest ways in

T I G - March 1987, VoL 3, no. 3

iTERMINAL ~LYCOSYI.ATION i

a - o T a_o~o-D-mm-Am 61¢

. - _~=~-s :.o,.~,,~. ~ ~_._..*-n ~ ~ _ ~ 0 - ~ ® []

CMPNAN ,P' • CMPNAN 6 P f C a -

GDPFuc[o(I,~]Fuc-TI |

I~-£L--I-- o.~. ~ _ ? _ o -'°-R

ig. 2. Terminal glycosylation of N-linked carbohydrates. The reactions ~t lead to corOlaion of biantennary complex carbohydra~s are zmmarized. Similar reactions are known to occur on branched, ~ected and hybrid moietie~S~mbols are the same as for Fig. I except r~t R ffi Gn-(Fnc)Gn-Am; GalNAc, 0 = N-ace~tgalactosamine; rAN, • = N-~tylaeurmninic ac'id and NGN, ~ = . N- lycolflncuraminic acid. The reac6ons km~m to be blocked in ammal ,,il glycosylation muta~zts are indicated by a slash and a dreled number phile transferase activities shown to be increased are des~mated by a umber in a square. All mutants with these properties, have been reuim~y reeiewed~6"Z° e~cOt for those desipmted m reactions 20 and 2 t2efs 11 and 12, reepedively). Mutants affeaed in pvoteoglycun ~thesis have also been described 13.

A--a--O'~o__ R a-,~.-m- o...e_ R fJIPN6N NGN-T A-T--° / i~&-II--o"

UDPGelIBII,llGoI-T ®-A-m-o.. -~4]n~-u-°~ o-R Q_&4__o -''°-R (A.I~.,A--I --,D

which these novel cell lines might be exploited in the future.

Glycosylation pathways Before glycosylation mutants were isolated, the

reactions involved in carbohydrate biosynthesis were deduced from evidence that exogenous carbohydrates of known structure would act specifically in vitro as substrates for certain glycosyltransferases. On the basis of these studies, the structures of endogenous substrates were postulated and schemes for biosyn- Lhetic pathways were proposed. Although this apvroach has given valuable insights into carbohy- drates which will not act as acceptors for certain

reviews enzymes, the hazards of proposing in vivo pathways with this type of evidence were clearly brought to light by the analysis of GIcNAc-Tl-deficient CHO mutants (designated 8 in Fig. 1). These mutants accumulate the endogenous substrate for GIcNAc-TI which, to everyone's surprise, was Mans-GIcNAce-Asn rather than the expected Mans-GIcNAc2-Asn (Refs 16 and 17t. Further studies of these mutants identified two new enzyme activities, GIcNAc-TII (Ref. 18) and a- mannosidase l! (Ref. 19). It thus became obvious how powerful the genetic approach can be in identifying glyccsylation enzymes, transport or regulatory molecules whose existence is completely un- suspected.

In addition to being a useful source of enzyme activities in a simplified environment, giycosylation mutants are a source of novel carbohydrates that cannot be bought or easily made. Recessive mutants accumulate endogenous substrates while dominant mutants synthesize new carbohydrates not previously expressed by the cell. Both types of mutants have the potential for synthesizing structures that are al- together novel.

In the future it will be important to apply the genetic approach to understanding the factors involved in regulating glycosylation pathways. Construction of appropriate double mutants could be used to investigate the dependence of separate reactions on each other and to describe the biochemical conse- quences for carbohydrate biosynthesis of complex changes in the pathway. Reversion analysis is equally important. Second-site revertants should be par- ticularly helpful in identifying new molecules involved in carbohydrate biosynthesis. Finally, the observation that glycosyltransferase activities may be induced by mutation-like events in dominant mutants dramatically expands the number of glycosylation reactions that can be studied in a given cell line. It now seems feasible that genetic analysis in somatic cells will be possible for many, if not all, of the enzymes involved in carbohydrate biosynthesis.

Molecular g e n e t i c s - cloning glycosylat ion genes

To date the genetic analysis of glycosylation mutants has been confined to complementation studies. In the future, however, it will be important Lo map the different mutations, at least in the CHO

Table 1. Uses of glycosyla6on mutants

BiosynLk__~si9 of carbohydrates Biochemical Identification of: Glycosylation reactions

Endogenous substrates Transport molecules Regulatory molecules Novel carbohydrates Novel enzymes

Geneti~ [dentdicatzon of genes Generation of alleles Cloning by complemerttation Glycosylation gene expression Glycosylafion gene organization Glycosyl~Gc-~ gene ~X.~in~

Functions of catbohydratos lnlramolec~lar Activity, stability and compamuenta]Lzation of: Endogenous glycoconjugates

Wnl glycoproteins Transfectod glycoprotoins

l~¢rmo/ecu/ar Reco~ifion phenomena Membrane fusion Dilferentiation Transformation (Tumorigenesis) Colomzation (Metastasis) Adhesion Migration lnU~_eplhdm" strucUEe

r views genome t'or which a partial map already exists 2°. In addition it will be important to use the mutants to aid in cloning the genes dmt code for glycosylation enzymes (termed glycosylation genes) so that their organiza- tion and the factors affecting their expression can be defined.

The use of animal cell mutants to clone glycosylation genes by complementation might prove to be their most important contribution to an eventual under- standing of the functions of carbohydrates that change during development and differentiation. Although several glycosyltransferases from abundant tissues or fluids have been extensiveiy purified zl and may be cloned using classical approaches ~, by far the majority of glycosylation enzymes will be extremely difficult to purify because they are membrane-bound and present in catalytic amounts. However, genomic DNA or cDNA that encodes a glycosyltransferase activity can be used to transfect an appropriate mutant, thereby correcting the glycosylation pheno- type by complementation and opening the possibility of cloning the transfected gone by rescue in a cosrr~d or ~ library. Cloning by this approach has worked most notably for oncogenes and for a DNA repair gene =. Two glycosylafion.defective phenotypes have been corrected by DNA transfection of animal cells ~ '~ and yeast glycosylation enzymes have also been cloned by complementation 26.

A modified transfection technique using Polybrene and dimethylsuiphoxide shock has been shown to give improved transfection frequencies with CHO cells ~7. The changes in lectin-resistance pattern caused by the expression of particular glycosyltransferases are well- characterized in CHO cells e-e and therefore it should be possible to use lectins to select or screen for the expression of a transfected glycosyltransferase activity. In certain cases, monoclonal antibodies may also be used to screen for surface expression of a new carbohydrate structure zs. Thus if the mutants fulfil their potential, glycosylation genes encoded in human, mouse or any other type of DNA should be clonable by this approach.

Funct ions of carbohydrates in differentiat ion and development

Once cloned genes are available, it will be possible to examine the function of carbohydrates by an entirely new approach. For example, it is thought that the key enzyme in generating the mouse stage- specific antigen SSEA-1 at the late 8-cell stage of embryonic development is an oc-(1,3)-fucosyltrans- ferase. Once this enzyme is cloned the function of the SSEA-1 determinant during development could be examined by introducing the gone for the enzyme early (e.g. at the 2-cell stage when it is known that lactosamine-~erminating glycoconjugates are present) or by introducing a plasmid encoding antisense DNA to inhibit synthesis of the enzyme at the late 8-cell stage. In this way, insights m! 3 the relationship of SSEA-1 expression to the course of development should be obtained. Since the SSEA-1 antigen and other c~a'bohydrate antigens also appear following cellular transformation l'zs, a similar approach could be taken to examine the effects of prohibiting expression of specific glycosyltransferases in tumor cells.

The expression of new transferase activities by

TIG - - March 1987, VoL 3, no. 3

dominant glycosylation mutants (Fig. 2) is very reminiscent of the appearance of similar activities during development and differentiation 1. A key question, therefore, is whether the enzyme inductions in both instances are the result of altered gene expression and, if so, what changes in the genome are responsible for this phenomenon. For example, is the molecular basis of enzyme induction operating in LECI1 and LEC12 CHO mutants that express SSEA- 1 (Ref. 6) identical with the mechanism operating in vivo to generate SSEA-1 in embryos or in tumors? If similar mechanisms are responsible for the appearance of SSEA-I, the dominant mutants will provide excellent material with which to define the regulation of carbohydrate synthesis at the molecular level. Even if the mechanisms differ, study of the genomic changes in dominant glycosylation mutants should at the least provide insight into mechanisms of gone regulation in mammalian cells.

Intramolecular funct ions Glycosylation mutants offer the great advantage of

synthesizing glycoconjugates with specifically altered carbohydrate moieties. Animal viruses, when grown in a glycosylation mutant, express the altered carbohydrates on viral glycoproteins, allowing the functional role of these carbohydrates to be studied in terms of virus infectivity, stability and the ability to perform fusion reactions. Similarly, any glycoprotein for which the gene has been cloned can be tailored for carbohydrate content by transfection of the cloned gone into an appropriate mutant. This approach might become wry important in the production of clinically- relevant glycoproteins such as interferon or growth factors so that they possess optima] activity, stability and serum half-life.

Glycosylation mutants are also useful 10r investi- gating the effects of altered carbohydrate content on the expression of endogenous g[ycoproteins (e.g. receptors for hormones or growth factors). However, it must be kept in mind that alterations in the rate of synthesis of a receptor or a change iA1 its intracellular compartmentalization might be due to indirect effects of the new carbohydrates expressed by cellular glycoconjugates that interact with the molecule of interest.

h t termolecular functions CeU-surface properties

One of the fascinathlg observations made with glycosylation mutants is that marked changes in the array of carbohydrate structures expressed at the surface of cultured ceils have no apparent affect on their viability. Glycosylatinn mutants therefore represent a system for exploring the variation in carbohydrate structures that may be tolerated by a cell and used to generate recognition markers during cell and tissue differentiation. Their variety of cell surface carbohydrate phenotypes make the mutants excellent cells with which to define the e]dstence and specificities of carbohydrate-binding proteins (e.g. plant and animal cell lectins) and with which to explore the consequences of carbohydrate alterations on membrane fusion (e.g. animal viruses or myoblast fusion), cell adhesion, cell morphology, cell migration and cell differentiation.

TIG - - March 1987, VoL 3, no. 3

For example, certain lectin-resistant myoblasts are unable to fuse to form myotubes, thus implicating particular carbohydrate structures in this complex membrane event. In contrast, F9 teratecarcinoma cell glycosylation mutants with reduced expression of SSEA-1 are able to differentiate in culture, suggesting that this carbohydrate determir.ant is not critical to the formation of certain endoderm cells (reviewed in Ref. 6). In these differentiating culture systems both positive and negative findings shed light on potential functions for carbohydrates in vivo.

Tumorigenesi.~ Another interesting role for carbohydrates that has

been studied using glycosylation mutants ~s their apparent involvement in the tumorigenic or metastatic properties of a cell. Several groups have shown that specfific glycosylation mutations expressed in different tumor cell lines correlate with a reduction in tumorigenicity and/or a reduced ab~ty to metastasize ~. This is an important result because only certain carbohydrate changes gave the effect and because it establishes the feasibility of obtaining tight correlations by detailed genetic analysis. The factors involved in generating a tumor or in inducing metastasis (often to a specific location) are extremely complex, so that the ability to apply genetics to this problem is a great advantage. For example, when independent mutants, independent revertants and appropriate double glycosylation mutants were compared, only one ClIO glycosylation mutation, instead of the three indicated by a pilot study, was found to correlate completely with reduced tumorigenicity in nude mice 3°. The next challenge will be to determine the

mechanisms by which carbohydrates might cause alterations in tumorigenicity. Much excitement will ensue if it turns out that a particular carbohydrate structure is directly responsible for any of the complex steps that lead to tumor formation or metastasis.

Coneludingremarks Glycosylat/on mutants will continue to provide

avenues for determining the structureYfunction relationships of the carbohydrates associated with glycoconjugates in mammal/an cells. In the future, it is expected that DNA transfection techniqnes ~ be increasingly exploited for obtaining gZycoproteins with specific carbohydrate structures and cloning glyco- sylation genes. The molecular genetics of the genes that code for glycosylation enzymes will be a fascinating study that should unfold in the next decade°

Acknowledgements The author's laboratory is supported by grants from

the National Cancer Institute, The American Cancer Society, and the Irma T. I-lirschl Trust. Thanks are extended to Margaret Kieli~n, William Chaney, Daniel Howard and Sandra Sallustio for comments on the manuscript.

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1268-1275

P. Stanley is at tke Albe~ Einstein College of Medidns, Deparlment oJ Cell Biolo~,, Bronx, N Y 10461, USA.

Reviews scheduled for forthcoming issues of Trends in Genetics

Dorsal-ventral embryonic pattern genes of Drosophila,

by Kathryn V. Anderson The geneti¢~ of body scent,

by E. A. Boyse, G. g. Beauchamp and If. Yamezaki

Recombination and the concerted evolution of the murine MHC,

by J. Geliebter and S. G. Nathenson The evolution of mammalian sex chromosomes and dosage compensation- clues from marsupials and monotremes,

by Jennifer M. Graves Precision and orderliness in splicing,

by M. Aebi and C. Weissmann Ontogeny of T-cell receptor expression,

by W. Born