Several Galactosyltransferase Activities Are Associated with Mouse Chromosome 17

SEVERAL GALACTOSYLTRANSFERASE ACTIVITIES ARE ASSOCIATED WITH MOUSE CHROMOSOME 17

KIYOSHI FURUKAWA,**' STEPHEN ROTH* AND JANET SAWICKI?

*Department of Biology, University of Pennsylvania, and +Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 Manuscript received May 20, 1986 Accepted July 24, 1986

ABSTRACT Indirect evidence suggests that some major histocompatibility complex (MHC) proteins are glycosyltransferases. No sequence or mapping information is avail- able for transferases, although ganglioside variations in mice are linked to the H-2 complex on chromosome 17, and one galactosyltransferase activity on mouse sperm varies with T/t complex genotypes, also on chromosome 17. In the present experiments, diploid and trisomy 17 mouse embryos were assayed for four different galactosyltransferase activities. The same preparations were assayed for isocitrate dehydrogenase (ld-1, chromosome I) and glyoxalase-1 (Glo-1, chromosome 17). Galactosyltransferase specific activities in trisomy 17 embryos are almost 1.5 times higher than in diploid embryos. The correlation between galactosyltransferase activities and chromosome 17 dosage indicates that the structural or regulatory gene for these enzymes are located on chromosome 17.

LYCOSYLTRANSFERASES transfer sugars from activated donors to an G exceedingly wide variety of acceptors. Several lines of evidence suggest a relationship between glycosyltransferases and the products of the major histocompatibility complex (MHC) family of genes (ROTH 1985). First, like MHC products, some transferases have intercellular recognition functions (ROTH, MCGUIREand ROSEMAN197 1; SHUR 1982). Second, when carefully purified, glycosyltransferases appear to exist in structurally related groups (NAGAI, MUENSCHand YOSHIDA1976; NACAI et al. 1978a, b; FURUKAWAand ROTH 1985). Third, because each different glycosidic linkage is thought to be cata- lyzed by a different transferase (BEYERet al. 1981), the number of different transferases must be very large. Fourth, two murine transferase activities vary according to changes in the T/t and H-2 regions (SHUR and BENNETT 1979; HASHIMOTOet al. 1983), both of which are located on chromosome 17. Fifth, polyclonal and monoclonal antibodies raised against purified galactosyltransferases cross-react with immunoglobulins (WILSON et al. 1982; PODOLSKYand ISSELBACHER1984) that share sequence homology with MHC proteins (HONJO 1983). Gene dosage effects on enzyme specific activities have been demonstrated

Current address: The Institute of Medical Science, The University of Tokyo, Tokyo 108, Japan.

Genetics 114 983-991 November, 1986. 984 K. FURUKAWA, S. ROTH AND J. SAWICKI for a number of mouse aneuploids (EPSTEIN et al. 1977; COX, TUCKERand EPSTEIN1980). Because no transferase has yet been mapped, a study of galactosyltransferase specific activities in diploid and trisomy 17 mice was initiated. Trisomy 17 embryos can be produced, although they die at about 10-12 days of gestation (GROPP 1978; EPSTEINet aE. 1982). TO test the relationship between chromosome 17 and glycosyltransferase specific activities, l l-day embryos were karyotyped and assayed for galactosyltransferases, as well as isocitrate dehydrogenase (chromosome I) (HENDERSON1965; HUTTONand RODER- ICK 1970) and glyoxalase-1 (chromosome 17) (MEo, DOUGLASand RUNBEEK 1977). The data show that four galactosyltransferase specific activities vary in proportion to chromosome I7 dosage.

MATERIALS AND METHODS Materials: UDP-[6-'H]-GaI (16.3 Ci/mmol) was purchased from Amersham (Arling- ton Heights, Illinois), and its purity was monitored periodically by high-voltage paper electrophoresis in 1% sodium tetraborate. Human a1 -acid glycoprotein was generously donated by M. WICKERHAUSER(American Red Cross Blood Services Laboratories, Be- thesda, Maryland). All other reagents were purchased from Sigma Chemical Company (St. Louis, Missouri). Mice: Litters containing diploid and trisomy 17 mouse embryos were generated by mating mice with the normal complement of 40 acrocentric chromosomes to mice carrying two different Robertsonian fusion metacentric chromosomes; the two fusion chromosomes carry chromosome 17 as a common arm (WHITE et al. 1974; GROPP, KOLBUSand GIERS 1975). Thus, when B6D2F1 mice are crossed to mice doubly heterozygous for two Robertsonian translocation chromosomes-Rb(2.17)11 Rma and Rb(8.17)l Iem-the two metacentric chromosomes often segregate together during meiosis. The result is a trisomy 17 embryo with cells that carry 39 chromosomes, including the two metacentric chromosomes, and a total of 41 chromosome arms. Although Rb(2.17) 11 Rma/Rb(8.17)1 Iem females and males are fertile, and can be mated to B6D2F1 mice of the appropriate gender to produce embryos, males doubly heterozygous for a different pair of Robertsonian translocation chromosomes- Rb( 16.17) 7Bnr/Rh(8.17)1 Iem-are sterile. Some of the trisomy 17 embryos and their diploid littermates were derived, therefore, from mating Rb( 16.17)7 Bnr/Rb(8.17)1 Iem females to B6D2F1 males. Females bearing trisomy 17 and diploid embryos were dissected at 11 days of gestation, using plug day as day 0. Litters containing diploid and trisomy 8 embryos were generated in a similar manner. Rb(8.12)22 Lub/Rb(8.17)1 Iem females were mated to B6D2F1 males; pregnant females were dissected at 9 days of gestation. B6D2F 1 mice and homozygous translocation stocks were obtained from Jack- son Laboratories, Bar Harbor, Maine. Chromosome analysis: To determine the karyotype of each embryo, amnionic membrane cells were arrested in metaphase, and their chromosomes were subsequently spread (EVANS,BURTENSHAW and FORD 1972). Each membrane was incubated in Dul- hecco's Modified Eagle's Medium (I g of dextrose/liter) containing 5% fetal calf serum and 0.0 15 pg vinblastin (Velban, Grand Island Biological Company, Grand Island, New York) per milliliter for 6 hr at 37" in humidified air containing 5% CO2. Membranes were then incubated in 0.56% KCI for 8.5 min, ethanol/acetic acid (3:l) for 30 min and 60% acetic acid for 5 min. Chromosomes were then spread on glass slides pre- warmed to 50-70". Sonication and homogenization: Dissected embryos were suspended in 300 pl phosphate-buffered saline containing 0.1% Triton X-1 00. Embryos were either homogenized with a glass pestle fitted to a plastic centrifuge tube or sonicated 3 X 90 sec at 0" with a Fischer Sonic Dismembrator at one-third energy level. The method of embryo dis- ruption had no effect on any of the enzyme assays. MOUSE CHROMOSOME I7 TRANSFERASES 985 Galactosyltransferase assays: Galactosyltransferase assays were conducted as described in detail (FURUKAWAand ROTH1985) in a final volume of 50 p1 containing the following reagents: 23 PM UDP-['HI-Gal (390 mCi/mmol), 3 mM 5'-AMP, 0.1% Triton X-100, 15 mM MnC12, 20 mM 2[N-morpholino]ethane sulfonic acid, pH 6.5, 10 pl of embryo homogenate, and 250 pg asialo-, agalacto-al-acid glycoprotein (AsAgAGP), which was prepared as described (FURUKAWAand ROTH 1985). All galactosyltransferase assays on trisomy 17 embryos, and their littermates were incubated for 3 hr. All transferase assays on trisomy 8 embryos and littermates were incubated for 6 hr. After terminating the reactions, the incubation mixtures were subjected to high voltage electrophoresis on borate-impregnated paper, and the galactosylated product was determined by excising the dried origins and counting them in a liquid scintillation counter. To assay Gal transfer to other acceptors, these were added in place of AsAgAGP, and at the following concentrations: asialo-ovine submaxillary mucin (AsOSM), 250 fig; GlcNAc, 10 mM; GalNAc, 10 mM; ganglioside GM2, 8 mM. For transferase assays using AsOSM, which was prepared as described (FURUKAWAand ROTH 1985), GalNAc, and GM2, manganese concentrations were 10 mM. Transfer to endogenous Gal acceptors was determined by omitting added acceptor. Isocitrate dehydrogenase assay: Isocitrate dehydrogenase activity was determined colorimetrically by incubating 10 PI of embryo homogenate in a total volume of I ml containing 1 mM isocitrate, 5 mM MnCI2, 0.5 mM NADP, and 0.02% bovine serum albumin in 50 mM sodium cacodylate, pH 7.5, at 37" for 5 min. After termination with EDTA, the a-ketoglutarate produced was reacted with 2,4-dinitrophenylhydrazine and converted to the chromogen phenylhydrazone in an alkaline solution (AMADORand WACKER1965). Glyoxylase-1 assay: Glyoxalase-1 activity was determined spectrophotometrically by measuring the increase in A240 nm in 1.5 ml of a solution containing 10 ~1 of embryo homogenate, 0.03% reduced glutathione and 0.04% methylglyoxal, based on the method described by RACKER(1 95 1). UDP-Gal hydrolysis: To determine the extent of hydrolysis of the sugar donor, UDP-Gal, pyrophosphorylase activity was determined by the method of SPIK,SIX and MONTREUIL(1 979). After incubation for 3 hr, galactosyltransferase assay mixtures were subjected to paper chromatography in pyridine:ethylacetate:water:acetic acid (5:5:3:1) for 24 hr. For standards, authentic UDP-Gal, Gal-1-phosphate and Gal were included. The areas to which Gal-1-P and Gal migrated were cut from the paper and counted in a liquid scintillation counter. Protein was determined by the method of LOWRYet al. (1951) using bovine serum albumin as a standard.

RESULTS Characteristic metaphase chromosome spreads from diploid and trisomy 17 amnionic membranes are shown in Figure la and b. The presence of two metacentric chromosomes is indicative of trisomy. The karyotype of each embryo from five litters was determined. At least five unambiguous spreads were examined for each embryo. Figure 2 shows the incorporation of Gal to AsAgAGP and AsOSM by trisomy 17 and diploid embryo homogenates as a function of time. Although transfer to AsAgAGP is not linear after 1 hr, trisomy 17 specific activities are consistently higher than diploid activities. Gal transfer to AsOSM is linear with time for up to 3 hr, and is also higher in trisomy 17 embryos, although the transfer rates by both embryo types are much reduced compared to those for AsAgAGP. The results of enzyme assays of 11 diploid and eight trisomy 17 embryos 986 K. FURUKAWA, S. ROTH AND J. SAWICKI

FIGURE I.-Metaphase chromosome spreads prepared from diploid (a) and trisomy I7 (b) amnionic membranes. Metacentric chromosomes are indicated by the arrows. The presence of two metacentrics (2,17 and 8,17) and 41 chromosome arms is indicative of trisomy 27. are presented in Table I. Galactosyltransferase specific activities toward four acceptors are higher in trisomy 17 embryos than in diploid embryos, with an overall ratio of trisomy:diploid specific activities of 1.48. This value is consistent with the presence of three copies of chromosome 17 in aneuploid embryo cells, as compared to two copies in diploid cells. As expected, the specific activity of glyoxalase-1, encoded by chromosome 17, is 1.5 times higher in trisomy 17 embryos than in diploid embryos. In contrast, the specific activity of isocitrate dehydrogenase, encoded by chromosome 1, is identical in both types of embryos. The elevation of the transferase and glyoxalase activities occurs in trisomy 17 embryos generated by parents carrying two different pairs of Robertsonian fusion metacentric chromosomes. It remained possible, however, that elevated transferase specific activities did not reflect increased dosage of chromosome 17, but rather the delayed development characteristic of some trisomies. To test this possibility, trisomy 8 embryos, which display developmental delay and mortality patterns similar to those of trisomy 17 embryos (GROPP 1982), were generated and assayed iden- tically. As shown in Table 2, galactosyltransferase, glyoxalase and isocitrate MOUSE CHROMOSOME I7 TRANSFERASES 987

0 1 2 3 lncubat ion Time (h) FIGURE2.-Galactosyltransferase specific activities in trisomy 17 and diploid mouse embryos. Galactosyltransfer to AsAgAGP (circles) and AsOSM (triangles) by trisomy 17 (solid symbols) and diploid (open symbols) mouse embryo homogenates. Transfer to endogenous acceptors occurs at about 0.08 nmol Gal/hr and has been subtracted from all points.

TABLE 1 Isocitrate dehydrogenase, glyoxalase-1 and galactosyltransferase specific activities in 1 l-day trisomy 17 and diploid mouse embryos

Embryo ploidy Trisomy/ Enzyme activity Trisomy 17 Diploid diploid pa Isocitrate dehydrogenase (A4001 0.91 f 0.13 (7)b 0.93 f 0.1 1 (8) 0.98 NSc mg protein-min) Glyoxalase-1 (A240/mg protein- 0.45 * 0.03 (4) 0.30 f 0.02 (4) 1.50 CO.01 min) Galactosyltransferases (nmol Gal transferred/mg protein-3 hr) to: AsAgAGP (250 fig) 4.28 f 0.43 (7) 2.88 5~ 0.40 (11) 1.49 CO.001 AsOSM (250 pg) 1.03 k 0.15 (6) 0.73 f 0.13 (10) 1.41 CO.01 GIcNAc (10 mM) 1.31 f 0.04 (5) 0.85 * 0.08 (4) 1.54 <0.001 GalNac (10 mM) 0.91 * 0.15 (7) 0.62 f 0.12 (10) 1.47 C0.05 Ganglioside GM2 (8 mM) ND~ (4) ND (4) “p values refer to comparisons between the mean specific activities of trisomy and diploid embryos. Values given are means f standard deviations (n). NS, Not significant. ND, Not detectable. dehydrogenase specific activities in trisomy 8 and diploid embryos do not differ significantly. 988 K. FURUKAWA, S. ROTH AND J. SAWICKI TABLE 2 Isocitrate dehydrogenase, glyoxalase1 and galactosyitransferase specific activities in 9day trisomy 8 embryos and diploid littermates

~ Embryo ploidy Trisomy/ Enzyme activity Trisomy 8 Diploid diploid

Isocitrate dehydrogenase (A400/mg 0.41 2 0.10 (2)" 0.50 f 0.07 (4) 0.82 protein-min) Glyoxalase-I (A240/mg protein-min) 0.28 2 0.01 (2) 0.34 f 0.03 (4) 0.82 Galactosyltransferases (nmol Gal transferred/mg protein-3 hr) to: AsAgAGP (250 pg) 2.55 2 0.42 (2) 2.58 f 0.11 (4) 0.99 AsOSM (250 pg) 0.56 f 0.10 (2) 0.54 2 0.09 (4) 1.04 Values given are means 2 standard deviations (n). Because the galactosyltransferase assays were performed with UDP-Gal concentrations that are very close to the typical K,,, values for these enzymes in other systems (FURUKAWAand ROTH 1985), it is possible that the trisomy- diploid differences are not due to higher transferase specific activities, but to lower rates of sugar nucleotide degradation in the trisomy 17 embryos. To test this possibility, incubation supernatants from trisomy 17 and diploid embryos were subjected to paper chromatography in solvents that separate UDP- Gal from its two breakdown products, Gal-I-phosphate and Gal. UDP-Gal hydrolysis is very low (<0.5%) and is similar in diploid and aneuploid embryos. The virtual absence of sugar donor hydrolysis is probably due to the presence of the. competitive inhibitor, 5'-AMP (MOOKERJEAand YUNG 1975), in all transferase assays.

DISCUSSION Galactosyltransferase specific activities toward four different acceptors are 1.5 times higher in trisomy 17 mouse embryos than in diploid littermates. Previously, when structural genes encoding enzymes have been mapped to a particular chromosome, aneuploids for that chromosome, either monosomics or trisomics, display the expected dose-dependent specific activities (EPSTEINet al. 1977; Cox, TUCKERand EPSTEIN 1980; GARTLERand RIVEST 1983). Be- cause galactosyltransferase specific activities increase in a dosage-dependent manner with respect to chromosome 17, it is likely that structural or regulatory genes for these enzymes are on chromosome 17. Glyoxalase-1, known to be encoded by a gene on chromosome 17, shows an appropriate dose response when the specific activities of diploid and trisomy 17 embryos are compared. On the other hand, isocitrate dehydrogenase, encoded by a gene on chromosome 1, shows no activity variations in response to different numbers of chromosome 17. Identical assays performed with trisomy 8 embryos indicate that the elevated transferase specific activities occur only with increased dosage of chromosome 17. The present assays were limited by the very small size of 1 1-day, trisomy 17 MOUSE CHROMOSOME 17 TRANSFERASES 989 mouse embryos, each of which was assayed separately, and each of which contain about 1.5-2 mg of total protein. The resulting need for high-specific activity UDP-Gal necessitated UDP-Gal concentrations that were less than op- timal. As a result, Gal transfer becomes nonlinear very quickly, as seen in Figure 2. Another problem that arises from suboptimal sugar donor concentrations is the effect of sugar donor degradation by hydrolases that may be present. In these assays, 5’-AMP was an apparently effective inhibitor of sugar donor hydrolysis. These data are consistent with a recent hypothesis (ROTH 1985) that predicts that glycosyltransferases are, or are the immediate evolutionary precursors of, the MHC proteins, which in the mouse are encoded by genes on chromosome 17. It remains possible, of course, that the genes encoding the galactosyltransferases assayed here are on chromosome 17, but at a site distant from the MHC, and that they are entirely unrelated to the MHC genes. A definitive answer to the question of MHC/transferase relatedness must await a comparison of the amino acid sequences of homogeneous transferases with those of MHC proteins, or a comparison of the nucleotide sequences of genes known to encode transferases with those of MHC genes. Recently, for example, SHAPERet al. (1986) have isolated a possible bovine milk galactosyltransferase cDNA clone by screening an expression library immunologically. Because antibodies against particular transferases appear to cross-react with other classes of transferases (DESCHUYTENEERet al. 1985; ROY CHOWDHURYet al. 1985), it is possible that the screening antibody might have reacted with a host of other transferases as well as with the specific galactosyltransferase in question. Despite this caveat, and despite the species difference, the predicted amino acid sequence shows scattered regions of homology with human immunoglobulin su- perfamily proteins like secretory component, ,62 microglobulin and immunoglobulin heavy chain hinge region. A catalytic function for MHC proteins could explain the autoimmune dis- eases that associate with particular HLA specificities, and would bring a new dimension to studies of MHC-restricted immunological responses.

This research was supported by grants HD 15044 (to S.R.) and HD 16760 (to J.S.) from the National Institutes of Health, and by grant PCM 81-18625 (to S.R.) from the National Science Foundation.

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