The microvillar hydrolases: A model group of membrane proteins

166 THE M I C R O V I L L A R HYDROLASES: A MODEL GROUP OF M E M B R A N E PROTEINS

GILLIAN M COWELL D e p a r t m e n t of Biochemistry C The P a n u m Institute University of C o p e n h a g e n , Blegdamsvej 3 C o p e n h a g e n N, D e n m a r k

Introduction

Epithelial cells that are destined to perform absorptive functions, such as the intestinal enterocyte and the kidney proximal tubule cell, possess a polarized plasma membrane where the apical domain is specialized into a microvillar, or brush border, membrane. This membrane is endowed with a large number of ectoenzymes, predominantly peptidases and glycosidases, which are responsible for the final degradation of protein and carbohydrate into the constituent amino acids (or dipeptides) and monosaccharides that can be internalized by the uptake mechanisms of the cell. L-3 No other cell type musters a similar battery of plasma membrane hydrolases, but most of the enzymes are not microvillar specific a s s u c h . 4 Table 1 lists a few examples of some well-characterized microvillar enzymes. Over the past years, microvillar enzymes have been a consistently attractive subject for many research groups not only because of their important physiological function but also as a model group of proteins for more general studies on how polarized cells maintain two separate plasma membrane entities. This short review presents the more recent advances in our knowledge concerning the structure and biosynthesis of microvillar enzymes, stressing the common themes in nature's design of this important group of proteins.

Structure

The first major break-through in the study of the microvillar membrane and its enzymology was the development of a method for isolating whole brush borders. 5 Later, methods for preparing vesiculated microvilli were developed 6'7 and the first proteinasesolubilized enzymes were purified and characterized. Intact, amphiphilic microvillar enzymes, however, proved hard to purify by conventional chromatographic procedures, owing to close similarities in their overall physico-chemical properties. It took specific antibodies and immunoadsorbent chromatography s to achieve this goal which in turn paved the way for studies on the amphiphilic molecular structure. The first surprising feature of microvillar enzymes to be revealed was their apparent anchoring via a Nterminally located segment. This so contradicted the orthodox view, stemming from

Table 1

Some examples of Microvillar enzymes and their biological activity Enzyme

EC No

Physiological substrate(s)

Aminopeptidase N

3.4.11.2

Oligopeptides with a neutral N-terminal amino acid

Aminopeptidase A

3.4.11.7

Oligopeptides with an acidic N-terminal amino acid

Dipeptidyl peptidase IV

3.4.14.5

Oligopeptides with Pro or Ala as the penultimate N-terminal amino acid

Endopeptidase-24.11

3.4.24.11

Neuropeptides containing internal hydrophobic residues

Sucrase-isomaltase

3.2.1.48-10

Sucrose, isomaltose, maltose (~(1---~ 4) and e~(1 --> 6) bonds)

Maltase-glucoamylase

3.2.1.20

Maltose (or(1 ~ 4) bonds)

Lactase-phlorizin hydrolase

3.2.1.23-63

Lactose ([3 bonds)

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167 studies on spike proteins of enveloped viruses that Macnair and Kenny, 9 presenting their data for dipeptidyl peptidase IV, humbly asked if it be heresy to postulate the existence of an anchor near the N-terminust Today, of course, it is fully legitimate to hold this view as it has been recognized that membrane proteins can be associated with the lipid bilayer in many, often very complicated, ways. The similar overall molecular size of corresponding proteinase and detergent solubilized forms of microvillar enzymes gave birth to the idea of a 'stalked' integral membrane protein as a common structural denominator for this group of proteins. This concept has since been elaborated by more refined methods, most notably by electron microscopy of reconstituted enzymes1°-13 and lately, by cDNA-deduced sequencing of the primary structure. 14'15 The models, shown in Fig 1, are based on reconstitution experiments and incorporate the structural features that today are considered characteristic for microvillar enzymes. Four different domains can be recognized: A globular portion and a junctional segment, both situated at the extracellular side of the plasma membrane; a transmembrane segment and a cytoplasmic segment. The microvillar enzymes illustrated are homodimeric with the subunits most commonly held together by noncovalent forces. The globular portion carries the catalytic function and, as indicated above, comprises the major part (>95%) of the total molecular mass which varies from less than 100 kDa up to 250-300 kDa. For the peptidases, there are only two globular domains per functional enzyme molecule, in agreement with the notion that they each only contain one type of catalytic activity. The glycosidases have four catalytic domains. For sucrase-isomaltase, the sucrase portion of the molecule has no anchor of itself but is connected to the membrane via isomaltase. The existence of sucrase and isomaltase as two separate functional domains on the molecule supports the view that sucrase during evolution arose by a partial gene duplication of isomaltase,3 an idea that has been further strengthened by the high extent of sequence homology (41%) between the two subunits. 12 Although still unproved, an analogous explanation for the molecular composition of maltase-glucoamylase seems likely. Notice, however, the different ways in which the subunits of sucrase-isomaltase and maltase-glucoamylase are organized, the latter characteristically projecting in a butterfly-like fashion with the outer subunits far apart. The junctional segment consists of a single stretch of the polypeptide chain and its function is probably to provide a flexible link between the catalytic head group and the supporting membrane. It is generally the most vulnerable domain of the molecule being susceptible to cleavage by a wide range of proteinases. The length of the junctional segment varies from 2 nm for endopeptidase-24.11 and 2.5 nm for dipeptidyl peptidase IV to 5 nm for aminopeptidase N, and interestingly, there is good correlation between the length of the segment and the release of the head group by proteolytic cleavage.16 The membrane-anchoring segment likewise consists of a single chain of about 20, largely hydrophobic, amino acids that span the bilayer only once. In addition to its permanent role as an anchor, the membrane-spanning segment provides the signal function for

1 I

Figure 1

2

3

4

I

Tertiary and quaternary structure of microvillar enzymes Models of four different microvillar enzymes, based on electronmicroscopy of reconstituted proteins. 1, sucraseisomaltase; 2, maltase-glucoamylase; 3, aminopeptidase N; 4, dipeptidyl peptidase IV. Scale: Bar represents 5 nm

BIOCHEMICAL EDUCATION 15(4) 1987

168 membrane translocation during biosynthesis (discussed below). The cytoplasmic tail of microvillar enzymes is only a few amino acids long. For other types of plasma membrane proteins such as receptors, the cytoplasmic domain has been shown to be essential for their function, for instance in protein phosphorylation and internalization of ligands. Its limited size on microvillar enzymes probably reflects its minor importance here, but the presence of basic amino acids in this region of the molecule may prevent the polypeptide from slipping through the bilayer. Hypothetically, the cytoplasmic tail could act as a sorting signal during biosynthesis. The models presented here only intend to provide a picture of how microvillar enzymes are organized and show to what extent individual enzymes may vary within a common structural theme. Interesting exceptions exist, though. Thus, alkaline phosphatase and trehalase probably share some of the overall features but instead of a hydrophobic peptide anchor, they are linked to the membrane by covalent linkage to phosphatidylinositol. 17.18

Synthesis and Intracellular Transport

Subcellular site of synthesis Eukaryotic membrane protein synthesis principally occurs in one of two locations: in the cytosol on 'free' ribosomes or on ribosomes attached to the rough endoplasmic reticulum (RER). Different lines of evidence strongly argue that microvillar enzymes originate from the RER. Thus, microsomal membranes (containing vesiculated RER), added to cell-free translation mixtures are capable of processing (glycosylating) the primary translation products. Furthermore, pulse-chase labelling experiments show that the earliest detectable forms of microvillar enzymes are firmly associated with intracellular membranes. According to the classical 'signal' hypothesis,L9 this implies a mechanism of membrane insertion that is intimately coupled to polypeptide synthesis: The presence of a hydrophobic, N-terminal pre-sequence enables a translocation across the endoplasmic membrane and thus a de facto sequestering of the nascent polypeptide from the cytosol into the cisternal lumen of the R E R (Fig 2). Interestingly, signal-cleavage could not be demonstrated for microvillar enzymes, 2° supporting the notion that their N-terminal anchors are in fact permanent (non-cleaved) signal sequences, as had earlier been proposed, z1'22 Direct proof of this has recently been provided by the cDNA-deduced primary sequence of two microvillar enzymes. ~4'15 Another unusual property of the membrane translocating process has been revealed by a so called synchronous translation experiment. 23 Here, it was observed that up to 25% of aminopeptidase N (approx 250 amino acids) can be synthesized on free ribosomes without loss of the ability to translocate the endoplasmic membrane, indicating that the timing between initiation of polypeptide synthesis and association with the membrane need not be all that strict (Fig 2). This view has recently been confirmed by studies on other types of membrane proteins. 24 2

3

4

./

N

Figure 2

Mechanisms for cotranslational membrane translocation Protein synthesis is initiated by free ribosomes (1). Ribosomal attachment to the endoplasmic membrane can occur either at an early or late stage during chain elongation (2). The nascent polypeptide conceivably can cross the membrane linearly or via a loop (3), resulting in anchorage by the C-terminal end or N-terminal end, respectively (4). The sequence of events shown in the upper panel may represent the mechanism for biosynthesis of many viral membrane proteins, but the scenario, shown in the lower panel, is the one most likely for microvillar enzymes

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169

Transport to the microvillar membrane Pulse-chase labelling experiments, combined with subcellular fractionation, the use of inhibitors of various cell functions, and, more recently, the application of immunogold-electronmicroscopy,25-26 are the principal techniques whereby the route of transport, leading from the RER to the microvillar membrane, has been studied. In accordance with the 'membrane flow' theory of P a l a d e F the membrane-confined pathway passes through the Golgi complex (Fig 3), an organelle known to play a central role in the biogenesis of many types of membrane and secretory proteins. 28'29 However, disappointingly little is known for certain about the post-Golgi transport. The once attractive idea of the basolateral plasma membrane acting as a transit station between the Golgi complex and the microvillar membrane has now largely been abandoned in favour of a direct delivery to the apical surface. Thus, immunogold electronmicroscopy has failed to detect microvillar enzymes in the basolateral plasma membrane but localized them in smooth, 100 nm vesicles in the apical cytoplasm. 26 The antimicrotubular agents colchicine and vinblastine interfere with the post-Golgi transport, 3°-32 suggesting that microtubules, possibly by a tread-milling mechanism, 33 provide the microvillar-directed dynamic force for these putative transport vesicles. However, a further elucidation of the post-Golgi transport awaits a purification and further characterization of such vesicles.

PROTEOLYTIC PROCESSING

COLCHICINE, VINBLASTINE MONENSIN, SWAINSONINE

e

TUNICAMYCIN,

C

A.O -

SPERMINE

Figure 3

*

CARBOHYDRATE

ss,.G

MEMBRANE INSERTION, HIGH MANNOSE GLYCOSYLATION

Intracellular transport and molecular processing of newly synthesized microvillar enzymes A schematic drawing of the flow of newly synthesized microvillar enzymes in a polarized cell from the RER to the Golgi complex and-- via putative transport vesicles -- to the apical surface. Along this route of transport, a highly ordered series of molecular modifications takes place. The arrows to the left indicate the subcellular target sites for various inhibitors of transport and processing

Kinetics of the intracellular transport A noteworthy characteristic of the biosynthesis of microvillar enzymes is the time required for the transport to their site of function; it generally takes from 1 to 3 h in the different pulse-chase labelling experiments reported. 2° Considering the short half-life of microvillar enzymes in the intestine, 34 this means that they may spend a very high proportion of their life in an unproductive state of transport. The long transport time possibly reflects a complex series of events necessary to sort microvillar enzymes from newly synthesized proteins with other destinations, such as endoplasmic, Golgi, lysosomal, basolateral and secretory proteins, a process that needs be accomplished with a high degree of perfection in order to maintain the compartmentation of an eukaryotic cell. 28 Kinetically, the pre- and post-Golgi stages of transport can be distinguished owing to the Golgi-associated processing which markedly changes the Mr of microvillar enzymes (from transient to mature forms, see below). Since no intermediate stages between these two molecular forms can normally be detected, their pool size - - and thus the time spent

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170 traversing the Golgi complex itself-- must be comparatively small. Several workers have observed that different microvillar enzymes are transported to the cell surface at varying rates. 35-37 Furthermore, the evidence indicates that the pre-Golgi leg of the transport is where the rates differ while the post-Golgi transport occurs at approximately the same rate. These differences are probably based on a varying accessibility to the cell's transport/sorting system. They could speculatively be taken as evidence for the existence of a receptor-mediated transport but can likewise be explained by varying rates of dimerization of the newly made microvillar enzymes, an event that may well be of importance for intracellular transport and sorting. 38

Processing

N-linked glycosylation

During their assembly in the R E R and the subsequent intracellular transport, the naked, primary translation products of microvillar enzymes invariably undergo a complex series of modifications, commonly referred to as 'processing' which in turn transforms them to mature enzymes. The most general type of modification of membrane and secretory proteins that originate from the R E R and one that has been observed for all microvillar enzymes so far studied is the attachment of high mannose oligosaccharides to susceptible asparagine residues on the nascent polypeptide chain 39 (N-linked glycosylation). This carbohydrate, however, does not remain intact; even before completion of the polypeptide chain, glucose residues are removed by specific glycosidases and in a highly ordered fashion, this trimming is continued during passage through the Golgi complex where also sugars of so called complex type (galactose, sialic acid, fucose) are added by glycosyltransferases. Despite its ubiquituous character, it has been difficult to ascribe a general role to N-linked glycosylation. Nevertheless, its importance for microvillar enzymes has been assessed by use of various inhibitors that block this processing at well-defined stages. Tunicamycin, which totally blocks the attachment of high mannose oligosaccharides, led to the formation of non-glycosylated forms which failed to appear in the microvillar membrane. 4° The non-glycosylated forms could only be detected when the proteinase inhibitor leupeptin was simultaneously present, indicating a rapid degradation of naked enzyme molecules. Castanospermine, blocking the initial removal of glucose residues, predictably gave rise to non-trimmed high mannose forms of increased Mr, but did not prevent intracellular transport to the Golgi complex and subsequent expression in the microvillar membrane. 41 Again, but less drastically than with tunicamycin, castanospermine caused a leupeptin-sensitive degradation of newly synthesized microvillar enzymes. Finally, swainsonine, which blocks the action of the Golgi-associated mannosidase II, resulted in the formation of partially processed (so-called 'hybrid') forms of aminopeptidase N. Interference at this late, posttranslational stage only marginally affected the transport to the microvillar membrane and it did not seem to destabilize the newly made enzymes. 32"42 In summary, we can therefore conclude that attachment and cotranslational trimming of high mannose oligosaccharides are both important in conferring molecular stability to the nascent microvillar enzymes whereas the modifications taking place during passage through the Golgi complex are of minor importance in this respect. There are no indications that N-linked glycosylation plays any role in targeting the enzymes to the microvillar membrane in a way that it does for enzymes destined for the lysosomes. It is noteworthy, however, that for one enzyme, sucrase-isomaltase, trimming and complex glycosylation is important for acquisition of biological activity whereas for aminopeptidase N and maltase-glucoamylase it is of no apparent relevance. 43

O-linked glycosylation The considerable difference in Mr (20-30 kDa) between the transient and mature forms of many microvillar enzymes strongly suggests that for at least some enzymes, N-linked glycosylation is not the only type of processing. O-linked glycosylation, a characteristic modification of gastrointestinal mucins, 44 is fairly widespread also amongst membrane proteins and some evidence for its occurrence during synthesis has been given. 2° Furthermore, rabbit aminopeptidase N has been shown to carry human blood group A determinants which are expressed by O-linked glycans. 45 One can speculate that O-linked carbohydrate, together with its N-linked counterpart, has a protective role for microvillar enzymes in their unhealthy working environment in the lumen of the gut amidst pancreatic proteinases. BIOCHEMICAL EDUCATION 15(4) 1987

171 Proteolytic cleavage It is well known that many intestinal microvillar enzymes are modified in situ by the action of pancreatic proteinases, but contrary to most hormones and other secretory proteins as well as lysosomal enzymes, the microvillar enzymes do not undergo a common type of posttranslational cleavage during intracellular transport. Exceptions to this are ~-glutamyl transpeptidase and lactase-phlorizin hydrolase which are both synthesized as precursors of higher Mr than the corresponding mature enzymes. For ~/-glutamyl transpeptidase, there is evidence that the precursor is cleaved into the heavy and light subunits of the mature enzyme at a pre-Golgi stage, but extracellular cleavage in the microvillar membrane has also been reported. 2° That the cleavage of the precursor thus may occur by different proteinases along the biosynthetic pathway suggests that it is of minor functional importance as, apparently, is the in situ fragmentation of other microvillar enzymes referred to above. In contrast to ~-glutamyl transpeptidase, the lactase-phlorizin hydrolase precursor is cleaved after acquisition of complex glycosylation but before arrival at the microvillar surface, that is, probably at the trans side of Golgi complex or during post-Golgi transport. 2° The cleavage is leupeptin-sensitive but is not essential for microvillar expression.

Regulation of Expression

The activity of intestinal microvillar enzymes increases during the pre- and early post-natal development and glucocorticoids are involved in their regulation. 46 For aminopeptidase N, comparable amounts of mRNA in adult and foetal intestine (as determined by cell-free translation) has indicated a control at a translational level in the enzyme's expression. 47 In the case of sucrase-isomaltase, on the other hand, mRNA levels (as determined by quantitative hybridization to a cDNA probe) correlated well with the changes in enzyme activity, indicating a transcriptional level of control. 48 These apparently contradictory results may be explained by the different methods used in so far as cell-free translation is a measure of the amount of translationally active mRNA whereas hybridization to cDNA is a direct measure of the total amount of mRNA, whether it being active or not. The divergence, however, could also reflect the difference in developmental pattern of the two microvillar enzymes. It is evident, however, that further work is necessary to obtain a more detailed and general view on how the expression of microvillar enzymes is regulated.

Concluding Remarks

In this review, I have aimed to convey the idea that the diversity of enzymic functions, characteristic of the digestive/absorptive surface of microvillar membranes, arose by relatively small variations of a common biosynthetic theme. It is likely that this view will soon become consolidated by an avalanche of primary sequence data, the fruit of the use of recombinant technology; thus the homology already discovered amongst some of the glycosidases predictably will be matched by homologies within the large contingent of peptidases. Until now, specific antibodies have been indispensable in the study of microvillar enzymes, both in situ and in the various subcellular locations they pass through en route. In the near future, DNA probes will allow us to look for the genes of microvillar enzymes within the cell nucleus and should be able to give us some insight into the way this interesting group of proteins are genetically organized.

Acknowledgements

Drs Hans Sj6str6m, Ove Nor6n and E Michael Danielsen are thanked for a critical discussion of the manuscript, The manuscript was kindly typed by Ms Asta Wieland Pedersen.

References

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