It is not known whether RG-I is synthesized onto existing HG or rather is synthesized independent of HG. If it is synthesized onto HG, it not known whether GAUT1 is that GalAT responsible for synthesizing the HG backbone region which would serve as a primer for RG-I backbone synthesis, or whether other GAUTs or other enzymes would perform this function.

It is also not known whether the alternating [^4)-a-D-GalpA-(1^2)-a-L-Rhap-(1^] backbone repeat is synthesized by a single glycosyltransferase containing both RhaT and GalAT activity, or whether, alternatively, the RG-I backbone is synthesized by a protein complex containing both a GalAT and a RhaT. If the backbone is synthesized by a complex, it is also not known whether GAUT1 or one or more of the GAUT1-related gene family members are part of the complex. To date, no RG-I-specific GalAT or RhaT has been reported.

5.4.10.1 RG-I:galactosyltransferase (RG-I:GalT)

RG-I synthesis requires at least eight galactosyltransferase (GalT) activities (Table 5.2). Probable p-1,4-GalT and p-1,3-GalT activities were originally identified in studies of mi­crosomal preparations from mung bean (375, 376) and more recently a mung bean p-

1,4- galactosyltranferase activity with a pH optimum of 6.5 was confirmed (377). Multiple galactosyltransferase (GalTs) activities have also been reported in particulate homogenates (378, 379) and solubilized enzyme (380) from flax (Linum usitatissimum L.). Detergent- solubilized flax microsomal GalTs transferred [3H]Gal from UDP-[3H]Gal onto exogenous RG-I-enriched and pectic p-1,4-galactan acceptors (381) to yield high molecular mass radiolabeled products. Surprisingly, the pH optimum for transfer onto lupin pectic p 1,4- galactan (i. e., pH 6.5) was different than the pH optimum for transfer of Gal onto an endopolygalacturonase-treated RG-I-enriched fractions from flax (i. e., two optima: pH 6.5 and 8.0) (381). Analysis of the products using RG-I-specific enzymes confirmed that the GalTs indeed added Gal onto RG-I (381), and thus, represented RG-I:GalTs. Furthermore, fragmentation of at least part of the product with p-1,4-endogalactanase demonstrated that at least some of the GalT activity represented p-1,4-galactosyltransferase (381). At pH 8, the GalT activity had an apparent Km of 460 ^M for UDP-Gal and characteristics consistent with a function in catalyzing the addition of galactose onto short galactan side branches of RG-I.

Microsomal membranes from potato suspension cultured cells have been shown to contain RG-I:p-1,4-galactosyltransferase activities that both initiate and elongate p-1,4- galactan side chains of RG-I (382). The potato RG-I:p-1,4-GalT activity in microsomal membranes had a pH optimum of 6.0-6.5 and produced a >500 kDa product using UDP — [14C]Gal and endogenous acceptor(s) in microsomal membranes. The product was frag­mented by endo-p-1,4-galactanase into [14C]Gal and [14C]galactobiose and into radiola­beled fragments between 50 and 180 kDa in size (382) when treated with the RG-I-specific rhamnogalacturonase A, an endohydrolase that cleaves the glycosydic linkage between the GalA and Rha in the RG-I backbone (383). The GalT activities in the microsomal membranes could be solubilized from the membranes using detergent (382) and the solubilized enzyme fraction was shown to contain at least two distinct GalT activities, one with a pH optimum of 5.6 that preferentially added Gal onto an ~1.2-MDa RG-I acceptor with a mole % Gal/Rha ratio of 0.7; and the other with a pH optimum of 7.5 that preferentially added Gal onto a 21-kDa RG-I acceptor with a mole % Gal/Rha ratio of 1.2. Neither activity could use RG-I acceptor containing lower Gal/Rha ratios, RG-I backbone without side chains, or galac­tan polymers or oligomers as acceptors, suggesting that the activities identified required recognition of the RG-I backbone and some existing Gal in a side chain (384). Interest­ingly, only the product synthesized onto the 21-kDa RG-I acceptor was digestible with a

1.4- p-endogalactase, suggesting that either the Gal transferred onto the larger RG-I acceptor was of a linkage other than (3,1-4, or that the length of the galactan side chain synthesized was less than three, the minimum size recognized by the 1,4-p-endogalactanase. The RG-I:p-

1.4- GalT that elongates the (3-1,4-side chains of RG-I was shown, by subcellular organelle fractionation and protease sensitivity experiments, to be a Golgi-resident protein with its catalytic site facing the lumen of the Golgi (385), a location consistent with its role in pectin synthesis. No gene for any RG-I galactosyltransferase has been reported.

More recently, (3-1,4-GalT activity in mung bean detergent-treated microsomal mem­branes was identified that transferred up to eight galactosyl residues in a (3 -1,4-linkage onto the non-reducing end fluorescently labeled exogenous (1^4)-p-galactooligosaccharide ac­ceptors (386, 387). Of the galactooligosaccharide acceptors used, i. e., degree of polymeriza­tion (DP) of 3-7, the galactoheptaose was a most effective acceptor although acceptors of DP 4-6 also functioned. The fluorescently labeled trimer was not active. Interestingly, fluo­rescently labeled RG-I backbone oligosaccharides of DP 5-7 were also not active, suggesting that the GalT activity identified could not add Gal onto oligosaccharide RG-I backbone regions. The (3-1,4-galactan:p-1,4-GalT activity had a pH optimum of 6.5 and apparent Km of 32 pM for UDP-Gal and 20 pM for the fluorescently labeled galactoheptaose (386).