ARTICLE |
Correspondence to: James R. McMillan, Dept. of Dermatology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo 060-8638, Japan. E-mail: jrm57@med.hokudai.ac.jp
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Summary |
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Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the 3-chain first globular domain) that was measured at 35.40 ± 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the ß3-chain globular domain IV) that measured 45.20 ± 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 ± 6.28 nm from the PM. The laminin 5
2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 ± 1.65 nm from the keratinocyte PM. Taken together with previous
2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27° from the horizontal lamina lucida (LL)/LD border and propose that the
2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions. (J Histochem Cytochem 51:12991306, 2003)
Key Words: electron microscopy, hemidesmosome, extracellular matrix protein, laminin 6
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Introduction |
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LAMININS are a large family of extracellular trimeric glycoproteins involved in cellmatrix adhesion in the epidermal basement membrane. At least 15 isoforms have been identified, each comprising three specific polypeptide chains (-, ß-, and
-chains). Laminin isoforms are determined by the specific chain composition that enables laminins with different properties to be expressed in a cell-specific manner (
6ß4 (
6ß4 integrin (
Previously, immunogold electron microscopy (IEM) has shown that laminin 5 is present in the lower lamina lucida (LL) (2-chain antibody to the lower LL. However, this technique relies on good antibodytissue penetration, which is not always possible with antibodies attached to colloidal gold particles or when inaccessible areas, including the basement membrane LD, are labeled. In such cases, postembedding labeling is a more reliable technique. We have reported, using postembedding methods, that laminin 5 localizes to the lower LL/LD border with the majority of labeling in the upper lamina densa (LD), including the
2-chain labeling that was restricted to the mid-LD (
Laminin is believed to be involved in epidermal cell adhesion, polarity, and migration, and to act as a developmental signal for hemidesmosomal assembly (6ß4 integrin is believed to be located within globular domains of the
3-chain (
3-chain perpendicular to the LD with the five globular domains in closest apposition to the keratinocyte (see Fig 3a and bi). We aimed to determine and compare a more precise distribution and position of laminin 5 globular domains of the
3-chain with a ß3-chain domain-specific antibody and antiserum against the whole molecule using an IEM technique.
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Materials and Methods |
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Skin Samples
Samples of normal skin from nonspecialized sites (abdomen and thigh from two individuals n=2; n=4 in total) were obtained at routine surgical procedures and were used for postembedding immunogold electron microscopy.
Indirect IEM
Samples of normal human skin were cryofixed and processed for postembedding IEM according to the methods previously described (
The sections were pre-incubated in buffer containing PBS with 5% normal goat serum (NGS), 1% bovine serum albumen (BSA), and 0.1% gelatin. The sections were then incubated with the mouse monoclonal antibody BM165 directed against the first globular (G1) domain of the laminin 5 3-chain (1:50) (
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Primary antibodies or antisera were all diluted in PBS buffer containing 1% NGS, 1% BSA, and 0.1% gelatin and incubated at 37C for 2 hr. The sections were then washed in a drop of PBS buffer four times for 5 min each and placed on a drop of secondary linker antibody, again diluted in PBS buffer (for 2 hr at 37C). Secondary antisera against mouse anti-rabbit IgG or rabbit anti-mouse IgG (Dako; Ely, UK) were diluted 1:500. Then the sections were incubated with a third antibody, 5-nm gold-conjugated labeled goat anti-rabbit or goat anti-mouse antibody (Biocell; Cardiff, UK) diluted 1:500 in Tris-buffered saline (TBS) for 2 hr at 37C. The sections were washed twice in TBS buffer and twice in distilled water (5 min each). After staining with 15% alcoholic uranyl acetate (15 min) and lead citrate (1 min), the sections were observed in a Hitachi H-7100 transmission electron microscope (Tokyo, Japan).
Immunogold Distribution Assessment
The techniques for ultrastructural measurements were similar to those previously performed (
Only non-obliquely (perpendicular) sectioned areas of BMZ were included in the assessment with clearly defined LL and LD. The basement membrane beneath melanocytes or damaged areas was excluded from this study. The distance from the plasma membrane to the center of each gold particle was measured and recorded (Table 1). The mean distance of each gold particle from the plasma membrane was calculated in µm ± SEM (Table 1) using the Minitab statistical package (Minitab; University of Pennsylvania, State College, PA). Gold particles that appeared clumped or associated with any deposit were also excluded. Each gold particle was also scored for its location immediately beneath a hemidesmosomal plaque and the percentage of labeling beneath the HD for each antibody or antiserum was calculated and expressed as a percentage of the total basement membrane labeling (Table 1). Three statistical analyses tests were performed using the Minitab software package. Two-way analysis of variance, Student's t-test, and the MannWhitney confidence interval test were performed (at 95% and 99% confidence intervals) to compare the respective mean antigen distances.
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Results |
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Normal Control Skin Labeling
Immunogold labeling for laminin 5 antibodies and antiserum showed strong signals in the basement membrane (Fig 1af). Immunogold particles (5 nm in diameter) were observed along the LL/LD boundary for the first 3-chain globular domain-specific antibody (BM165). This labeling showed an extended distribution, slightly higher in the mid-LL (Fig 1a and Fig 1b) in contrast to the ß3-chain antibody (K140) that showed relatively sparse labeling, the majority of which was restricted to the LD but also extended to the LL/LD border (Fig 1c and Fig 1d). The polyclonal antiserum showed abundant labeling over the majority of the LD but also extended to the lower quarter of the LL (Fig 1e and Fig 1f) in a similar fashion to BM165 (Fig 1a and Fig 1b). The average values for both laminin 5 antibodies and the antiserum were calculated from measurements of the distances from gold particles to the plasma membrane (Table 1). The
3-chain first globular domain antibody was located some 10 nm above the ß3-chain (35.4 ± 2.20 nm from the plasma membrane vs 45.2 ± 3.60 nm) and the polyclonal antiserum was located between these two values at 43.4 ± 6.28 nm below the keratinocyte plasma membrane (Table 1).
The distribution of all laminin 5 labeling is represented in graphic form for BM165 (Fig 2a), K140 (Fig 2b), and polyclonal laminin 5 (Fig 2c). The 3-chain globular domain showed a more restricted distribution range (1575 nm) than both the ß3-chain (K140; Fig 1c and Fig 1d) and the polyclonal antiserum (Fig 1e and Fig 1f). Estimates of the size of laminin 5 come from measurements made from rotary shadowing experiments (
3- and ß3-chains and knowing the approximate dimensions of laminin 5, we used the Pythagorean theorem and simple triangle geometry to calculate the approximate angle of the long axis of laminin 5 relative to the LL (to approximately 27°) (Fig 3bii), as follows: right-angled triangle one side: a1 = 110 nm, b1 = 45 nm, c1 = 118 nm with the smallest angle
1 = 22°; right-angled triangle two sides: a2 = 117 nm, b2 = 10 nm, c2 = 118 nm with the smallest angle
2 = 5°.
1 = 5 +
2 = 22 = total 27°
The samples of normal skin used in this and previous studies have demonstrated that the majority (7588%) of laminin 5 labeling was restricted to immediately beneath the hemidesmosome plaque (Table 1). The two 3- and ß2-chain antibodies and one serum showed a remarkably similar percentage labeling associated with the anchoring filament complex beneath hemidesmosomes, ranging between 77 and 78% (Table 1). There was no difference in the laminin 5 labeling distance between the samples from different body sites. Statistical analyses revealed differences between BM165 and both K140 and the whole polyclonal laminin 5 antiserum (p<0.05) and a highly significant difference between BM165 and SE153 (p<0.01; Table 1). K140 and the whole laminin 5 polyclonal serum were different from the SE153 staining (p<0.05). There was no difference between K140 staining and the whole laminin 5 polyclonal serum (p>0.05).
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Discussion |
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We have demonstrated in detail the localization of specific 3 and ß3 domains and furthermore have suggested a new orientation of laminin 5 in the human basement membrane. The globular domains of the
3-chain of laminin 5 are localized to the lower quarter of the LL, closer to the epidermis than the majority of the laminin 5 molecule (using the
2-chain and polyclonal antiserum for reference).
2-chain short arm was measured at 56 nm from the plasma membrane (
3-chain epitope was mapped 38 nm from the plasma membrane (similar to our findings). We therefore hypothesize that laminin 5 is oriented with the
3-chain globular domains some 3540 nm from the keratinocyte plasma membrane. The
2-chain is even farther from the plasma membrane, in the lower LD. These data and previous reports (
3-chain long axis is obliquely aligned within the LL and LD.
Previous reports of the laminin 5 molecular shape using rotary metal shadowing of molecules isolated from cultured cells suggest that the 3-chain long axis is approximately 110 nm in length and that the length of the two ß3 and
2 short arms is between 60 and 70 nm (
3-chain of laminin 5 is at an angle of approximately 27° to the LD border, much more obliquely aligned than was previously thought (Fig 3). However, caution should be exercised in interpreting this data due to the potential flexibility of laminin 5 in vivo or the possibility that it exists in a shape other than the proposed cruciform shape as seen using rotary shadowing.
This hypothesis supposes that laminin 5 maintains a rigid cruciform structure but, as rotary metal shadowing studies can demonstrate, there may be parts of the laminin 5 molecule with considerable flexibility (3 or
2 domains that may be stably anchored to the hemidesmosomal integrin
6ß4 (Fig 1c and Fig 1d vs Fig 1a and Fig 1b). Taken together with the previous data (
2-chain labeling deeper within the LD (Table 1) and the ß3-chain labeling in the lower LL, this suggests that some molecular flexibility may be involved. Alternatively, laminin 5 may have a rigid structure but may pivot about one or more fixed points that may include the
3-chain
6ß4 integrin cell binding globular domains or about a separate collagen VII, nidogen or fibulin 2 binding domain (
The average LL width in the human epidermal basement membrane was measured at 4550 nm (
Various laminin forms are expressed in the epidermal basement membrane, including laminin 5 (3ß3
2) and laminin 10 (
5ß1
1), and some less abundant forms may also be present, including laminin 1 (
1ß1
1), laminin 6 (
3ß1
1), and laminin 7 (
3ß2
1), previously identified in amnion, as well as laminin 11 (
5ß2
1) (
3-chain data may include antibody measurements from laminins 5, 6, and possibly 7. The presence and relative expression levels of intact laminins 6/7 beneath normal epidermis have not yet been confirmed. However, from our data, particularly the narrow peak for the
3-chain (see Fig 2a), we suggest that either that laminin 6/7 is not abundantly expressed in normal skin or that it shares a limited
3 domain localization similar to laminin 5. The expression of laminin 6/7 was first identified in vitro and in amnion (
1 arm domains (
6ß4 integrin (
6ß4 in the LL space have yet to be measured, but estimates using other methods have suggested that the
6-subunit may extend as far as the mid-LL (
The short arms of the ß3- or 2-chain of laminin 5 interact with the noncollagenous (NC-1) fibronectin-like domains of collagen VII (
2-chain (56 nm) (data from
2-chains remain likely candidates for laminin 5-collagen VII interactions. The laminin 5
2-chain (domain IV) has also recently been implicated in binding of nidogen and fibulin 2 (
2-chain at the lowest position within the lamina densa would facilitate such interactions (
In conclusion, the position of the laminin 5 3 first globular domain within the lower LL and the relative position of the
2 and ß3 short arms in the LD suggest the need for a revised orientation of this molecule, which will help to shed new light on the organization laminin complexes and possible laminin interaction partners. Our findings, together with advances in antibody production and characterization, pave the way for detailed examination of other epidermal basement membrane antigens and their likely interactions.
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Footnotes |
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1 These data were presented in part at the Society for Cutaneous Ultrastructure Research meeting, June 2002, Limoges, France.
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Acknowledgments |
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Supported by a grant-in-aid of Scientific Research A (13357008, H.S.), a Health and Labor Sciences Research Grant (Research into specific diseases H13-02), by a grant from the Japanese Society for the Promotion of Science (JSPS, grant #00345, J.R.M.), by a grant-in-aid for JSPS fellows' research expenses (#00345) and by an award from the Japanese Society for Investigative Dermatology International Fellowship (Shiseido) (2000).
We gratefully acknowledge the technical support of Mr. H. Nakamura and Drs F. Iwao and H. Nakamura for supplying the normal control skin samples, and M. P. Marinkovich for the generous gift of antibodies and antiserum used in this study. We also thank Dr T. Masunaga for kindly providing the data from the 2 chain studies.
Received for publication January 28, 2003; accepted May 28, 2003.
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