MATERIALS AND METHODS Sources of proteins Laminin-1, in complex with nidogen-1 and its elastase fragment E10, were obtained from a mouse tumor basement membrane [5,25].. Mouse Engelbret
Trang 1Eur J Biochem 269, 431-442 (2002) © FEBS 2002
Domain IV of mouse laminin ñ1 and B2 chains
Structure, glycosaminoglycan modification and immunochemical analysis of tissue
contents
Takako Sasaki’, Karlheinz Mann’, Jeffrey H Miner, Nicolai Miosge® and Rupert Timpl'
'Max-Planck-Institut ftir Biochemie, Martinsried, Germany, Renal Division and Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA; Center of Anatomy, Department of Histology, University
of Gottingen, Germany
Domain IV, consisting of about 230 residues, represents a
particular protein module so far found only in laminin BI
and $2 chains Both domains were obtained by recombi-
nant production in mammalian cells They showed a
globular structure, as expected from electron microscopic
examination of laminins Fragment BITV was obtained as a
monomer and a disulfide-bonded dimer, and both were
modified to ~50% by a single chondroitin sulfate chain
attached to Ser721 of an SGD consensus sequence
Dimerization is caused by an odd number of cysteines,
with three of them having a partial thiol character
Whether both modifications also occur in tissue forms of
laminin remains to be established Fragment B2IV was
only obtained as a monomer, as it lacked one crucial
cysteine and the SGD sequence It required, however, the
presence of two adjacent LE modules for proper folding
Polyclonal antibodies raised against both fragments
showed no cross-reaction with each other and allowed establishment of Bchain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues This demonstrated a 5—25-fold lower content of B2 com- pared with B1 chains in various tissue extracts of adult mice Tissues derived from §2-deficient mice failed to react with the B2-specific antibodies but showed a twofold higher content of $1 than heterozygotes The antibodies to 2 showed broader tissue staining than reported previously, including in particular a distinct reaction with the extra- synaptic endomysium of skeletal muscle Immunogold staining localized both Bchains primarily to basement membranes of kidney, muscle and various other tissues Keywords: basement membranes; chondroitin sulfate; laminin domain; radioimmunoassay; recombinant pro- duction
Laminins represent a family of heterotrimeric proteins (ay)
which are mainly localized in basement membranes and
involved in cell—-matrix and various other protein interac-
tions Fifteen different isoforms are so far known, laminin-1
to laminin-15, based on the assembly of different «1 to 5,
BI to B3 and yl to y3 chains [1—3] These chains share a 600-
residue domain ILI which oligomerizes into a rod-like
coiled-coil structure forming the long arm of laminins The
N-terminal short arms and C-terminal G domains, however,
are composed of laminin-type LE, L4, LN and LG
modules, which form rod-like or globular structures in
various combinations [1,4] Most of these modules are also
shared by several other extracellular proteins such as the
proteoglycans perlecan and agrin A few other domains are
so far unique to laminins and include domain IV of the BI
and B2 chain Only BITV has so far been obtained as a
proteolytic fragment [5]
Ten of the laminin isoforms contain either the B[ or
B2 chain in combination with yl and one of the five « chains
Correspondence to R Timpl, Max-Planck-Institut fiir Biochemie,
Am Klopferspitz 18A, D-82152 Martinsried, Germany
Fax: + 49 89 8578 2422, Tel.: + 49 89 8578 2440,
E-mail: timpl@biochem.mpg.de
(Received 8 august 2001, revised 31 October 2001, accepted 7
November 2001)
[6-8] These two Bchains consist of about 1760 residues, have an identical modular structure, and show about 50% sequence identity [9,10] Their mRNAs are expressed at different levels in a large number of tissues [10,11], produced
by a variety of cultured cells, and, as shown by antibody staining, encode proteins found in various basement mem- branes [12-16] Their distribution can change during embryonic development, particularly in aorta, kidney and skeletal muscle The $2 chain was actually discovered in a search for proteins that are specific for neuromuscular synapses [9] and this restriction has been confirmed in subsequent studies of mouse [15] but not human tissues [17-19]
Little evidence is, however, available on potential func- tional differences between B81 and £2 chains Laminins containing either variant were shown to promote in an equivalent manner cell adhesion [6] and neurite outgrowth [7] These activities are attributed to the shared «chains and apparently not modulated by different Bchains The analysis of chimeric Bchains provided evidence that a 16-residue sequence close to the C-terminus of domain I is directing the synaptic localization of B2-containing laminins [20] A Leu-Arg-Glu sequence in a similar region of
62 chain was also claimed to provide a specific stop signal for neurons [21] This sequence seems, however, not to be active when installed in a native coiled-coil conformation [22] A particular role for the B2 chain was also emphasized
Trang 2by gene knock-out studies, which demonstrated in mice
distinct deficiences in neuromuscular synapses and glomer-
ular filtration [23,24]
In this study, we have used recombinant production of
domain IV in mammalian cells to explore further structural
and tissue differences of the B1 and B2 chain This revealed
glycosaminoglycan modification and disulfide-dependent
dimerization of BITV but not of B2IV, which could be
readily explained by sequence differences This also allowed
development of sensitive and specific immunological assays
for both Bchains that are useful for quantitative analyses
and examination of their distribution in tissues
MATERIALS AND METHODS
Sources of proteins
Laminin-1, in complex with nidogen-1 and its elastase
fragment E10, were obtained from a mouse tumor basement
membrane [5,25] Other recombinant fragments of mouse
laminins included «1IVa [26], B1 VI//V and B3VI/V (unpub-
lished) which were prepared by established procedures [27]
Construction of expression vectors and cell transfections
The templates used were a complete mouse Bl cDNA
(plasmid no 1609) provided by Y Yamada (NIDR,
Bethseda, MD, USA), and for ÿ2IV we used RNA from
embryonic mouse endothelial cells provided by A Hatzo-
poulos (GSF, Munich, Germany) The sense and antisense
primers for Bl were GTCAGCTAGCTAACGAGGTGG
AGTCCGGTTAC and GTCACTCGAGCTAAAGGCC
CGTCTGGTGAATCAAG, respectively, and for B2 GTC
AGCTAGCCCGTCCCTGTGACTGTGATG and GTC
ACTCGAGCTAGGCTTGACAGCCTGCAGGG,
respectively They were used for amplification by PCR and
RT-PCR, respectively These primers introduced at the 5’
end an Nhel site and at the 3’ end a stop codon followed by
an XhoI site to allow insertion into the episomal expression
vector pCEP-Pu containing the BM-40 signal peptide [28] A
Ser721Ala mutation was introduced into BITV by fusion
PCR [29].These vectors were used to transfect 293-EBNA
cells, and serum-free medium was collected from these cells
[28]
Purification of recombinant proteins
Conditioned medium was passed over a DEAE-cellulose
column equilibrated in 0.05 m Tris/HCl, pH 8.6, and eluted
with a linear 0-0.6 mM NaCl gradient The effluent was
monitored at 280 nm and by a carbazole assay [30]
Fragment BIIV was eluted at 0.2-0.3m and 0.3-0.4
NaCl, whereas most of fragment B2IV did not bind to the
column They were both purified on a Superose 12 column
(HR 16/50) equilibrated in 0.2 mM ammonium acetate,
pH 6.8 Fragment B2IV was then bound to a Mono Q
column in 0.02 m Tris/HCl, pH 8.0, and eluted with 0.03—
0.05 M NaCl
Characterization of protein structures
Cleavage with CNBr was performed in 70% formic acid
(24 h at 23 °C) in the dark under nitrogen The fragments
were separated by size-exclusion chromatography on a Superdex Peptide column (Amersham-Pharmacia; HR 10/ 30) in 0.1% trifluoroacetic acid/25% acetonitrile Further cleavage at enzyme/substrate ratios of 1 : 100 with trypsin
or endoproteinase Lys-C in 0.2 m NH4HCOs or with pepsin
in 0.1 M glycine/HCl, pH 1.9 (23 °C, 15 h) was followed by Superdex chromatography and/or HPLC on a Cg column [31] Differential alkylation with 4-vinylpyridine or iodoac- etate after partial and complete reduction with dithiothreitol
in 0.05 m Tris/HCl, pH 8.5, and 6 M guanidinium hydro- chloride/0.05 m Tris/HCl, pH 8.5, respectively, followed a previously used procedure [32] Digestion with chondroitin- ases ABC, AC or B (Sigma) followed a previous protocol [33]
Analytical methods Hydrolysis with 6 M or 3M HCI (16 h, 110 °C) was used to determine protein and hexosamine contents on an LC3000 analyzer (Biotronic) Edman degradation was performed on Applied Biosystems sequencers 473 and 492 following the manufacturer’s instructions The recovery of pyridylethylcy- steines and carboxymethylcysteines was estimated from the height of the respective phenylthiohydantoin derivatives in the corresponding sequencer runs Electron microscopy of rotary shadowed proteins [34], CD, and matrix-assisted laser desorption ionization MS [35] followed standard protocols Immunological methods
Generation of rabbit antisera, affinity purification of antibodies, ELISA titration, and radioimmunoassays fol- lowed established protocols [36] A radioimmuno-inhibition assay specific for recombinant mouse laminin fragment y1III3-5 using antiserum against laminin fragment P1 has been described [37] Immunoblotting using ECL Western- blotting reagents followed a previous procedure [38] Mouse Engelbreth-Holm-Swarm (EHS) tumor and tissues from adult mice and 18-day-old pups heterozygous for (B2 + /-)
or lacking ($2 —/—) the laminin B2 chain gene [23] were extracted with EDTA-containing buffers and then deter- gents; this was followed by digestion with bacterial colla- genase [39] These extracts were then used at dilutions of
1 : 10 and higher for radioimmuno-inhibition assays Immunofluorescence staining of frozen tissue sections folllowed previously used procedures [8,15] Gold particles (16 nm) were used to label affinity-purified rabbit antibodies
as described [40] Tissue sections on nickel grids were incubated for 15 min with NaCl/P;, pH 7.2, and then labeled gold diluted 1 : 200 for 16 h at room temperature Sections were rinsed with water, stained with uranyl acetate (15 min) and lead citrate (5 min), and examined with a Zeiss
EM 109 electron microscope Controls included colloidal gold alone or samples coated with goat anti-(rabbit Ig) IgG
or anti-(rat Ig) IgG and were all negative
RESULTS Expression and purification of recombinant domain IV from mouse laminin B1 and B2 chain
Domain IV of the B1 (position 541-771) and B2 (position 556-782) chains correspond to the central globular domain
Trang 3© FEBS 2002
NEVESGYYFTTLDHY IYEAEEANLGPGVVVVEROYIQDRIPSWTGPGFVR
ele Tl: TTP oelll de Pode [Ie I[IIIIIII |
EQVQPGYFRPFLDHLTWEAEAAQ - GQGLEVVERLVTNRETPSWTGPGFVR
VPEGAYLEFF IDNI PYSMEYEILIRYEPQLPDHWEKAVITVQRPGKIPAS
; |] :[|:: ::| :|:|::|:|:[TH:|:: | : : TTTT]
LREGQEVEFLVTSLPRAMDYDLLLRWEPQVPEQWAELELMVQRPGPVSAH
SRCGNTVPDDDNOVVSLSPGSRYVVLPRPVCFEKGMNYTVRLELPQYTAS
| |l:::[ [| [ |::[::|:|IIII:lL |[::l ::|:| |::
SPCGHVLPKDDRTQGMLHPNTRƑTVFPRPVCLEPGISYKL.KLKLTG-TG
700 708 5 720 730 738
GSDVESPYT- - FIDSLVLMPYCKSLDIFTVGGSGDGEVTNSAWETFOQRYR
se fe fe ostltdttele deel J] :: Lhe]
RAQPETSYSGLLIDSLVLQPHVLVLEMF - - - -SGGDAAALERRTTFERYR
CLENSRSVVKTPMTDVCRNIIFSISALIHOTGL
| Ị:: |:|::::| :::|:|JH:: : |
CHEEGLMPSKAPLSETCAPLLISVSALIYNGAL
Fig 1 Sequence alignment of domain IV (A) from mouse laminin B1
(top) and $2 (bottom) chains and domain structure of the laminin B1 and
62 chains (B) (A) Both sequences show 41% identity (bars) and 29%
conservative changes (dots) Cysteines are numbered I—S in Bl and 14
in B2, and carbohydrate attachment motifs (SGD, NYT) are high-
lighted in bold Asterisks mark corrections to the published genomic
sequence of mouse £2 [43] An arrowhead indicates the start of frag-
ment E10 [5] The numbering includes the signal peptides (B) Laminin
B1 and 62 chains have the same domain structures consisting of LN
and LE (circles) modules, domain IV, and a coiled-coil (cc) domain
ITI
in the short arm of these laminin chains [2] (Fig 1B) They
are of similar size and share 41% identical residues including
four out of five cysternes and 29% conservative replace-
ments (Fig 1A) Features unique to BIIV are single
potential acceptor sites for N-glycosylation and glycosami-
noglycan attachment, respectively Episomal expression
vectors were prepared for both domains in order to obtain
them as recombinant fragments from serum-free culture
medium of transfected human kidney 293-EBNA cells
Production and secretion of fragment BIITV occurred at high
rates (150-170 ngmL '-đay ”) as shown by radioimmuno-
assay (see below), whereas no protein could be detected after
transfection with the B2IV expression vector corresponding
to the sequence shown in Fig 1A This indicated a protein-
folding problem, which was overcome by adding two
laminin-type epidermal growth factor-like (LE) modules to
the N-treminus and C-terminus of B2IV (LES, position 523—
555; LE6, position 783-831, see Fig 1B), allowing produc-
tion of this longer fragment at a moderate level (5 ugmL
day’) This fragment will be referred to as B2IV
The initial purification of fragment BIITV on DEAE-
cellulose showed it to be present in about equal proportions
Laminin Bl and B2 chains (Eur J Biochem 269) 433
A 123 4 5 6
kDa
200 —
ll6 -
97
66
B 1 2 34 5 6
kDa
200 —
116
CO
66
4S — 3L
Zh
Fig 2 SDS/PAGE of purified recombinant fragments containing domain IV of B1 or $2 chain under nonreducing (A) and reducing (B) conditions Lanes were loaded with equal amounts of B1TV monomer (1), B2TV (2), BITV dimer (3), BITV substituted with chondroitin sulfate before (4) and after (5) digestion with chondroitinase ABC, and BIIV mutant S721A monomer form (6) Positions of calibrating proteins are shown in kDa on the left
in two pools eluted at 0.2-0.3 mM NaCl (pool 1) and 0.3- 0.4 m NaCl (pool 2) Pool 1 consisted mainly of a 34-kDa band, as expected for a BIITV monomer, and about equal amounts of a disulfide-bonded B1IV dimer (66 kDa), which could be separated by molecular sieve chromatography as shown by electrophoresis (Fig 2, lanes | and 3) Pool 2 coincided with a strong carbazole reaction for uronic acids,
Trang 4indicating the presence of a proteoglycan The correspond-
ing BIIV fragment was eluted in front of the BITV dimer
from a molecular sieve and showed a broad electrophoretic
band mainly in the range 70-110 kDa, which could be
converted into the monomer and dimer bands after
treatment with chondroitinase ABC (Fig 2, lanes 4 and 5)
Recombinant fragment B2IV did not bind to DEAE-
cellulose and showed, after molecular sieve chromatogra-
phy, a single band of 33 kDa (Fig 2, lanes 2), indicating its
monomeric nature and lack of glycosaminoglycan modifi-
cation Edman degradation of the purified recombinant
fragments showed a major single N-terminal sequence,
which was APLANEVESG for the £1 variants and
APLARPXD for B2IV, as expected from the vector
constructs The first four residues, respectively, were derived
from the foreign signal peptides introduced to allow
secretion of the proteins
Structural characterization of the fragments
The proper folding of the recombinant fragments was
examined by several methods Rotary shadowing followed
by electron microscopy showed small compact globular
shapes for BITV monomers and dimers (not shown) and
B2I1V (Fig 3B) The same globule was also observed for the
proteoglycan form of BITV, but with some of the particles
being connected to a long faint thread-like structure
(Fig 3A) As these threads were not observed for BI
monomers and dimers, they probably represent the glyco-
saminoglycan side chain Proteolysis studies of BIIV
monomer showed no significant change after trypsin
treatment (1-24 h), while elastase caused degradation to a
22-kDa fragment similar to the E10 fragment obtained from
elastase digests of laminin-1 [5] Fragment B2IV, however,
was readily cleaved within I-4 h by trypsin into fragments
smaller than 20 kDa but was resistant to endoproteinase
Ae
100nm
Fig 3 Rotary shadowing electron microscopy of chondroitin sulfate-
substituted fragment BITV (A) and fragment B2I1V (B) Bar: 100 nm
Glu-C (data not shown) CD spectra of the B1TV monomer showed a strong negative ellipticity at 209-220 nm, indicat- ing about 30% ahelix and only a small amount of
B structure (Fig 4) The 2IV structure had only half the a-helical content, which may have become obscured through the presence of two additional LE modules, which,
as shown previously, have only a small amount of secondary structure [41]
Hexosamine analysis of BITV monomer and dimer showed 5-6 residues of glucosamine and less than one galactosamine That Edman degradation of various prote- olytic peptides (see below) failed to identify Asn677, indicating full occupation of the single N-linked acceptor site The proteoglycan form of BITV showed the same glucosamine content, but a large increase in galactosamine content (48 residues), which corresponds to about 19 kDa
of a glycosaminoglycan chain Furthermore, digestion with chondroitinases ABC (Fig 2) and A,C but not by chon- droitinase B or heparitinase (not shown) yielded the BITV monomer and dimer bands, demonstrating the exclusive substitution by chondroitin sulfate The mean (SD) length
of the side chain was estimated from electron micrographs
of 30 particles to be 40 (+ 10 nm), which is in good agreement with a molecular mass of 20 kDa Mutation of Ser721 to Ala did not interfere with recombinant produc- tion of BITV monomers (Fig 2, lane 6) and dimers but prevented completely the modification by glycosaminogly- cans, consistent with the absence of any other strong acceptor site within the BITV sequence (Fig 1) Fragment B2IV lacked any of these substantial post-translational modifications, as determined by MS, which yielded a molecular mass of 34 369 Da, in good agreement with a mass of 34 357 calculated from the sequence
The odd number of cysteines in BITV indicated the presence of a free thiol group, which is probably responsible for dimerization The native BITV dimer was therefore first modified by partial reduction and pyridylethylation to protect the thiol followed by complete reduction under denaturing conditions and carboxymethylation Peptides were then generated by cleavage with endoproteinase Lys-C followed by cleavage with some other proteases and determination of the sequence around the five cysteines
0
E -5000
E
oO
»
Ø -10000
-15000
200 210 220 230 240 250
> (nm)
Fig 4 CD spectra of recombinant domain IV from laminin [1 (full line) and [2 (dashed line) chain.
Trang 5© FEBS 2002
(Cl to CS, Fig 1A) by Edman degradation The data
obtained showed complete carboxymethylation for Cl and
C2, while for C3, C4 and C5 there was also 20-25%
pyridylethylation Dimeric BIIV was also cleaved with
CNBr giving rise to three single-chain peptides starting at
the N-terminus and positions 609 and 677, respectively This
indicated C1l—C2 connectivity, which was confirmed by
Edman degradation of smaller pepsin fragments The fourth
CNBr peptide showed two sequences starting as expected in
front of C3 and C5 and should also contain C4 (Fig 1A) A
smaller pepsin fragment showed a connection between C3
and C5 Yet because of the variable thiol content of C3 to
C5 (see above), we cannot exclude the possibility that the
disulfide connectivity is flexible, and we suggest that partial
formation of C3—C4 and C4—C5 bridges occur as well
Immunochemical assays for domains B1IV and B2IV
and their application to the analysis of tissues
Many previous studies based mainly on 1mmunohistology
have shown a complex and in part overlapping expression
pattern of laminin B1 and £2 chains in adult tissues, during
embryonic development and in cultured cells (reviewed in
[2,16]) Several of the monoclonal antibodies against
32 chain were generated in mice and failed to react with
the corresponding mouse antigens [9] To circumvent these
limitations and to allow quantitative assays, we generated
rabbit antisera against recombinant mouse BIIV and B2IV
These antisera had high titers (half-maximal binding) at
dilutions of 1 : 4000 (anti-B1) and 1 : 20 000 (anti-B2) in
ELISA and radiotimmunoassays and did not cross-react
(titer less than | : 100) with the homologous (B2 or BI1IV)
antigen or recombinant N-terminal fragments B1VI/V and
B3VI/V of mouse laminin They were also clearly distin-
euishable in immunoblots of several biological samples,
with anti-B1 reacting mainly with a 220-kDa band and anti-
B2 with a 190-kDa band after electrophoresis under
reducing conditions (data not shown)
The antisera allowed the development of specific and
sensitive radiormmuno-inhibition assays, with half-maximal
inhibition being achieved at 0.1-0.2 nm BIIV and 2IV,
respectively (Fig 5) The BIIV assay was inhibited in nearly
identical manner by monomeric and dimeric BIIV and the
proteoglycan form and by laminin-1 («1 B1y1) derived from
the mouse EHS tumor (Fig 5A) Laminin-! fragment E10
showed a less steep and incomplete inhibition profile,
indicating the loss of some antigenic epitopes A more than
1000-fold excess of B2IV showed no inhibition (Fig 5A),
and the same was found for recombinant laminin fragments
BIVI/V, B3VI/V and o«1IVa (data not shown) Similarly, the
assay for B2IV could not be inhibited by a large excess of
B1IV (Fig 5B) Both assays could, however, be inhibited by
tissue extracts, as shown for mouse heart and kidney
(Fig 5), with inhibition curves similar in slope to that of the
reference inhibitors BIIV and B2IV, respectively Together
the data showed that these assays were highly specific and
suitable for the quantitation of B1 and 62 chain epitopes In
biological samples
The extracellular matrix of mouse EHS tumor has
previously been shown to consist exclusively of basement
membrane, and its major component, laminin-1, could be
solubilized in decent amounts (5 mg:g' wet tissue) by
extraction with EDTA and 6m guanidine [25] We now
Laminin B1 and B2 chains (Eur J Biochem 269) 435
dilution
100 A
ARR
Binding
0 —_
al L3 111101Ì a ng pg gyal L1 4 1110/01Ì L1 11/101Ì L1 gp pal
J Ỉ t Ỉ
se
.E
DO
.E
co
&
0 =
il kg pagal ag ga gyygyl ten gp ayy] L1 1111011 Ì bo pal
Inhibitor (nM)
Fig 5 Radioimmuno-inhibition assays specific for laminin B1 (A) and B2 (B) chains The assay consisted of 1 ng ‘I-labeled fragment BIIV monomer or B2I1V and fixed amounts of the corresponding antiserum Inhibitors used were B1TV monomer (A), chondroitin sulfate-substi-
tuted BITV (V), B2TV (A), mouse laminin-1 (©) and its E10 fragment
(L]) at the concentrations shown at the bottom EDTA extracts of mouse heart (@) and kidney (Ml) were used at the dilutions shown at the top
used this tumor and also mouse kidney and muscle for successive extraction with EDTA and detergents followed
by digestion with chondroitinase/heparitinase and bacterial collagenase, which solubilized nearly all of the tissue The extracts were analysed by the B1IV and B2IV assay and also
by an assay specific for the laminin yl chain [37] This demonstrated that 71-93% of the extractable laminin v1, BI and £2 chain were already solubilized by EDTA and detergent Furthermore, the content of extractable lami- nin-1 was calculated based on the yl and 61 chain assay to
be 5.5-5.8 mg:g ’ EHS tumor, in agreement with previous data A larger number of adult mouse tissues were then extracted with EDTA and detergent only and examined by these three assays (Table 1) A variable content of laminin
61 chain [17-422 pmol-(g wet tissue) '] and lower amounts
of B2 chain (420% of BI) were found As expected, the highest amounts of B1 chain were found in the EHS tumor, while the content of B2 chain did not exceed 0.2% The amounts of yl chain were within the usual range of
Trang 6Table 1 Contents [pmol-(g wet tissue) "| of laminin y1, B1 and B2 chains
in tissue extracts from normal adult and mutant (62 + /— B2 —/—-) mice as
determined by radioimmuno-inhibition assays Tissues were extracted
with EDTA-containing buffer and detergent and analyzed by assays
specific for recombinant fragments y1III3-5, BIIV and B2IV
analytical error (+ 20%) of such inhibition assays [36], in
most cases in good agreement with the sum of Bl and
B2 chains It served therefore as an internal control for the
quality of the data Tissue extracts of mice being deficient in
the laminin 62 chain [23,24] were used as further controls
and failed to inhibit the B2 assay (content less than
0.5 pmol-g ') Interestingly, these extracts showed a twofold
increase in the $1 chain content when compared with
heterozygous (82 +/-) controls (Table 1), indicating a
significant increase in biosynthesis or mRNA stability
Immunolocalization in tissues
The antiserum against recombinant mouse B2IV was tested
in immunohistochemical assays on a panel of mouse tissues
using indirect immunofluorescence (Fig 6) The antiserum
revealed a more widespread distribution of laminin 2 than
has previously been reported In the kidney, it was
previously shown to be confined to glomerular and vascular
smooth muscle basement membranes [9,12] Here, it was
detected in these basement membranes, but also in most
intertubular capillaries and segmentally in some tubular
basement membranes (Fig 6A) In skeletal muscle fibers, it
was concentrated at synapses but was also found extrasy-
naptically In peripheral nerve, it was found in the
perineurium, as previously reported [9,12], but also in the
endoneurium Blood vessels throughout skeletal muscle also
contained the B2 chain (Fig 6B)
In the small intestine, B2 was detected in smooth muscle
and blood vessels and at a low level in the epithelial
basement membrane of the crypts (Fig 6C) In lung, it was
present in basement membranes associated with bronchial
epithelium, bronchial smooth muscle, and alveoli through-
out the parenchyma (Fig 6D) In the retina, B2 was
detected in the inner limiting membrane, Bruch’s mem-
brane, and in capillary basement membranes, but was not
detected in the interphotoreceptor matrix or in other layers
of the retina (Fig E) In heart, B2 was found in cardio-
myocyte and vascular basement membranes throughout
(Fig 6F)
One reason for the discrepancies between published reports showing a restricted distribution of B2 and our results showing a more widespread distribution could be that the antiserum used here cross-reacts with another laminin chain in immunohistochemical assays To investi- gate this possibility, we immunostained tissues from Lamb2 mutant mice (Fig 7) These mice have a mutation that has been shown to prevent any accumulation of laminin 2 In basement membranes [23,24] No significant fluorescence was found on staining mutant tissues with the anti-B2IV serum, whereas tissues from a littermate control were well stained This demonstrates that the antiserum reacts with laminin B2 but with no other laminin chain and no other basement membrane component
Results from immunohistochemical assays using anti-B1 serum (data not shown) were mostly consistent with previously published reports showing a _ widespread (although not ubiquitous) expression pattern for laminin
Bl For example, in skeletal muscle, B1 was detected in the extrasynaptic muscle fiber basement membranes and in endoneurial basement membranes It was not observed at synapses or in the perineurium, sites where B2 is known to
be concentrated [12] In kidney, 81 was detected in all tubular basement membranes and in the glomerular mesangium In addition, it was detected at a low level in the glomerular basement membrane, a site where f2 is concentrated (Fig 6) Together with our B2 immunohisto- chemical results, these data suggest that many basement membranes contain both laminin B1 and £2 chains How- ever, in most cases one appears to be much more prevalent than the other
Affinity-purified antibodies specific for BITV or B2IV were also used for ultrastructural localization of both Bchains in adult mouse tissues by immunogold staining (Figs 8 and 9) This demonstrated for tubular and glomer- ular basement membranes of kidney a distinct reaction with both antibodies of about equal intensity (Fig 8A—D) and of basement membranes of collecting ducts, blood vessels and Bowman’s capsule In the heart, some weaker staining was observed in basement membranes adjacent to cardiomyo- cytes and endothelial cells (Fig 8E,F) and also in basement membranes of the endocardium and pericardium Staining
of skeletal muscle identified both B chains in endothelial and muscle cell basement membranes, whereas the adjacent interstitial region did not react (Fig 9A,B) A distinctly stronger reaction for B2 was observed in the synaptic clefts
of neuromuscular junctions compared with extrasynaptic regions (Fig 9C,D) and at myotendineous junctions Other basement membranes containing both f chains included the dermal-epidermal junction and testis, whereas the dermal connective tissue could not be stained
DISCUSSION Domain IV of laminin BI and B2 represents a globular structure in the central portion of the short arm region [1,2] and has not yet been characterized at the structural and functional level We have now obtained these domains as recombinant products from mammalian cells and demon- strated their proper folding by electron microscopy, CD, protease resistance, and immunochemical analysis The data also show that domain BIIV represents an autono- mous folding unit and was produced at high rate This was
Trang 7© FEBS 2002 Laminin BI and B2 chains (Eur J Biochem 269) 437
Fig 6 Immunohistochemical localization of laminin B2 chain in basement membranes of adult mouse tissues by antibodies against B2IV (A) In kidney, B2 is detected in basement membranes of glomerulus (g), large blood vessels (v), and most intertubular capillaries (arrows) Some segments of tubular basement membranes are also stained (B) In skeletal muscle, 82 is detected in basement membranes all around the muscle fiber (f) as well as the neuromuscular junction (arrowhead) In peripheral nerve (n), B2 is detected in both perineurial and endoneurial basement membranes (C) In small intestine, 82 is detected primarily in basement membranes of smooth muscle (sm), large blood vessels (v), and capillaries (arrows) Low levels
of B2 are detectable in basement membranes associated with crypts (c) (D) In lung, B2 is detected in basement membranes of the bronchial epithelium (e), bronchial smooth muscle (sm), and alveolar epithelium throughout the parenchyma (p) (E) In retina, 62 is detected in the inner limiting membrane (ilm), Bruch’s membrane (bm), and in capillaries (arrows) (F) In 3-week-old heart, B2 is detected in basement membranes surrounding all cardiomyocytes, large vessels (v), and capillaries (arrows) Bar, 100 um for A-C, F; 50 um for D; 75 um for E
surprisingly not the case for B2IV which needed two
additional LE modules for reasonable expression and
secretion Similar observations have been reported for the
N-terminal globular LN module (domain VI) of the
laminin ol chain, which could only be produced in
mammalian cells after it had been joined to a tandem of
four LE modules The latter, however, folded properly on its own [27] Another interesting observation is the relatively high content of ohelix in BITV and probably B21V A high helix content has so far only been demon- strated for the coiled-coil domains I and II of laminins [1,2], whereas other domains, such as those composed of
Trang 8Lamb2 +/
Lamb2 -/-
Fig 7 Antiserum to laminin B2IV does not react with basement membranes in Lamb2 mutant tissues Kidney (A and B) and skeletal muscle (C and D) from 3-week-old Lamb2 +/+ and -—/- littermates were stained with anti-B2 serum In the control, basement membranes throughout the kidney and skeletal muscle were stained Basement membranes at neuromuscular junctions (arrows in C) were more immunoreactive than were extrasynaptic basement membranes No immunoreactivity was detected in mutant tissues, demonstrating the specificity of the antiserum Neuromuscular junctions were identified by double labeling with rhodamine-o-bungarotoxin (C’ and D’) Bar, 100 um
LE or LG modules, essentially lack helical segments, as
shown by crystallographic analyses [41,42] This suggests
novel folding for domain IV, which can now be investi-
gated by X-ray analysis of recombinant BITV monomers
and dimers
Additional differences between domain IV of BI and
B2 chains are related to single amino-acid substitutions A
single serine within an SGDG consensus sequence was
shown by site-directed mutagenesis (S721A) to be partially
substituted by a 20-kDa chondroitin sulfate chain in both
the B1TV monomers and dimers This sequence is changed in
B2IV of rat, mouse and human [9,43] to SGGD and preceded by a four-residue gap (Fig 1), which may explain the failure to detect any glycosaminoglycan substitution in recombinant B2IV Yet an SGG site in perlecan domain V [44] and three SGD sites in domain I [33,45] can serve as acceptor sites for heparan sulfate/chondroitin sulfate, and more remote sequences may regulate their complete or partial occupation [45] Special features of such regulations may now be unravelled by site-directed mutagenesis of the corresponding regions in BITV and B2IV domains Our data also indicate that tissue laminins containing B1 chains may
Trang 9© FEBS 2002 Laminn BI and 2 chaIns (Er J Biochem 269) 439
Fig § Immunogold staining of laminin B1 (A,C,E) and j2 (B,D,F) chain in basement membranes of kidney and heart of adult mice Both antibodies reacted equally well with the basement membrane of a proximal tubule (A,B; arrows) and with the glomerular basement membrane (C,D; asterisks)
In the heart (E,F) both chains could be detected in the basement membranes of cardiomyocytes (arrows) as well as of endothelial cells of an adjacent capillary (asterisks) The capillary lumen (1) and the interstitial tissue between both basement membranes showed insignificant staining Bars, 0.25 pm
be, at least in part, converted into proteoglycans This is
underscored by conservation of the SGD sequence in the
human laminin 61 chain [46], whereas the laminin f chains
of Drosophila [47| and Caenrhabditis elegans (accession no
AAB 94193) lack this sequence and also differ in cysteine
patterns Preliminary unpublished studies to identify such
forms in tissue extracts have, however, failed so far and
indicated only a low level of substitution or a specific
restriction to a few tissue sites Yet we do not think that the
partial modification of BITV is an artefact of recombinant
production because a similar low rate of substitution of
recombinant perlecan domain V correlated well with a
comparable low rate of modification of two tissue forms of perlecan [44]
An extra cysteine (C3 at position 710) in domain BITV not conserved in B2IV was a candidate to explain the substantial dimerization of recombinant BITV We there- fore used stepwise reduction under nondenaturing and denaturing conditions as previously used to identify a single cysteine responsible for fibulin-2 dimerization [32] This provided clear evidence for a Cl—C2 connectivity within BITV but failed to identify unequivocally C3, C4 or C5 as being responsible for dimerization This could indicate the existence of various disulfide isomers, but we
Trang 10
Fig 9 Immunogold staining of laminin B1 (A) and B2 (B—D) in extrasynaptic and synaptic regions of skeletal muscle from adult mice Both antibodies (A,B) stained the basement membrane around myocytes (arrows) and the endothelium of a capillary (asterisks) The arrowhead indicates some staining close to a pericyte 1, Capillary lumen (C,D) shows some stronger staining for B2 of the synaptic cleft (asterisks) than for the extrasynaptic part (arrowheads) Bars, 0.25 jum
cannot exclude the possibility that they represent artefacts
of the treatments used Nevertheless, we also cannot
dismiss the possibility of the disulfide-dependent dimeriza-
tion of laminins in which domain BIIV participates Such
complexes between laminin-5 (a@383y2) and laminin-6
(a3Blyl) are known to exist in certain basement mem-
branes [48] or may be generated upon self-assembly of
laminins into networks, which occurs through their
N-terminal regions including the BI chain [49]
Laminin Bl and £2 chains have been localized by
immunohistology at the light microscopy level to a large
variety of tissues, but not necessarily to the same subana-
tomical regions, which has generated several controversial
observations (reviewed in [16]) We have now prepared
potent polyvalent antisera against the natively folded
fragments BITV and B2IV in order to re-examine some of
the previous data and to establish more quantitative and
sensitive assays The rabbit antisera obtained showed a high
specificity for Bl and 2 chains, respectively, as demon-
strated by ELISA, radioimmunoassay, and immunoblot
This was underscored by the failure to show any reactivity
of antibodies to B2 with tissues derived from f2-deficient
mice Specific radioimmuno-inhibition assays of high sen-
sitivity ICso = 0.10.2 nm) were developed and showed
both Bchains and equivalent levels of laminin y1 chains in
EDTA/detergent extracts of various mouse tissues The
content of BI chains in these extracts exceeded the content
of B2 by a factor of 5—20 The relative amounts of B2 were
particularly high in skeletal muscle, consistent, as shown here and previously, for developing and adult human muscle [17], by distinct staining of extrasynaptic areas of muscle basement membranes The radioimmunoassay data are also inconsistent with restriction to synapses, as shown
in rats by monoclonal antibodies [9,15], considering the low density of synaptic junctions (usually accounting for less than 0.1% of the total basement membranes) The radio- immunoassays also indicated a twofold higher content of B1 chain in lung and kidney of $2-deficient mice compared with littermate controls Such upregulation was _ previously predicted from immunostaining of kidneys, but it did not lead to functional compensation [24]
This is the first extensive examination of B2 distribu- tion in mouse tissues, most other studies having utilized rat, rabbit and human tissues Thus, there could be differences in $2 distribution among species However, restricted distribution of B2 in mouse kidney and skeletal muscle has been reported [15,23,24], and we have observed widespread expression of 2 1m rat muscle using anti-B2PV serum (data not shown) One possible explanation is that many laminin B2 monoclonal anti- bodies that have been produced may recognize a specific form of B2 found primarily in glomerular, synaptic, perineural, and smooth muscle basement membranes This could result from selective glycosylation, conforma- tional alteration, or association with a specific a chain Indeed, laminin «5 is also found in these basement